Blood drop patterns: Formation and applications

    Blood drop patterns: Formation and applications Ruoyang Chen, Liyuan Zhang, Duyang Zang, Wei Shen PII: DOI: Reference:

S0001-8686(15)30018-X doi: 10.1016/j.cis.2016.01.008 CIS 1621

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Advances in Colloid and Interface Science

Please cite this article as: Chen Ruoyang, Zhang Liyuan, Zang Duyang, Shen Wei, Blood drop patterns: Formation and applications, Advances in Colloid and Interface Science (2016), doi: 10.1016/j.cis.2016.01.008

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Blood Drop Patterns: Formation and Applications

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Ruoyang Chen1†, Liyuan Zhang1†, Duyang Zang2, Wei Shen1*

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1 Department of Chemical Engineering, Monash University, Wellington Road,

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Clayton Campus, Victoria 3800, Australia

2 Functional Soft Matter & Materials Group (FS2M), Key Laboratory of Space Applied Physics and Chemistry of Ministry of Education, School of Science,

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Northwestern Polytechnical University, Shaanxi 710129, China

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† Both authors are equally contributed to the paper. * Corresponding author: Wei Shen

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Department of Chemical Engineering, Monash University, Wellington Road, Clayton

Tel.: +61399053447

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Campus, Victoria 3800, Australia.

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E-mail: [email protected].

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ACCEPTED MANUSCRIPT Abstract The drying of a drop of blood or plasma on a solid substrate leads to the formation of

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interesting and complex patterns. Inter- and intra-cellular and macromolecular

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interactions in the drying plasma or blood drop are responsible for the final

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morphologies of the dried patterns. Changes in these cellular and macromolecular constituents in blood caused by diseases have been suspected to cause changes in the

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dried drop patterns of plasma and whole blood, which could be used as simple diagnostic tools to identify the health status of human and farming animals. However,

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complex physicochemical driving forces involved in the pattern formation have not been fully understood. This review focuses on the scientific development in

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microscopic observations and pattern interpretation of dried plasma and whole blood

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samples, as well as the diagnostic applications of pattern analysis. Dried drop patterns of plasma consist of intricate visible cracks in the outer region and fine structures in

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the central region, which are mainly influenced by the presence and concentration of inorganic salts and proteins during drying. Roles of the shrinkage of macromolecular

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gel and its adhesion to the substrate surface have been thought responsible to the formation of the cracks. Dried drop patterns of whole blood have three characteristic zones; their formation as functions of drying time has been reported in the literature. Some research works have applied engineering treatment to the evaporation process of whole blood samples. The sensitivities of the resulted patterns to the relative humidity of the environment, the wettability of the substrates and the size of the drop have been reported. These research works shed light to the mechanisms of spreading, evaporation, gelation and crack formation of the blood drops on solid substrates, as well as to the potential applications of dried drop patterns of plasma and whole blood in diagnosis. 2

ACCEPTED MANUSCRIPT Keywords: sessile drop evaporation; dried blood drop patterns; tension-caused

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cracking patterns; salt-induced drying patterns; the “coffee ring” effect; adhesion

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ACCEPTED MANUSCRIPT Contents 2

1. Introduction

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Abstract

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2. Background of the drop evaporation

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2.1. The “coffee ring” effect

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3. Plasma patterns

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3.1. Components in plasma

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3.2. Morphologies of dried plasma drop

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2.2. Current understanding of cracking patterns in colloidal drops

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3.3.1. Influence of inorganic salts

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3.3. Factors influencing plasma pattern formation

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3.4. Driving forces of cracking

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3.5. Mechanisms of the central pattern formation

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4. Whole blood patterns

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4.1. Components in whole blood

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4.2. Morphologies of dried blood drop

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4.3. Process of cracking pattern formation

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4.4. Factors influencing on blood pattern formation

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4.4.1. Influence of the relative humidity

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4.4.2. Influence of substrates

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4.4.3. Influence of the blood drop diameter and height

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4.5. Mechanisms of pattern formation

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5. Applications

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3.3.3. Influence of the plasma concentration and substrates

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3.3.2. Influence of the protein concentration

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5.2. Animal husbandry detection

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5.3. Limitations of applications

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5.1. Medical diagnosis

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6. The future perspectives

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Acknowledgements

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References

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1. Introduction

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The formation of complex patterns during drying of drops containing non-volatile matters

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is a common and interesting phenomenon in nature. In the past few decades, a variety of

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applications of dried drop patterns of biological liquid were reported in different research fields, such as archaeology [1, 2], DNA microarray printing [3, 4], medical practice [5] and

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forensic investigations [6-9]. Biological liquid is a complex system containing various components, including macromolecules and cells, suspended in a continuous aqueous phase.

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When a drop of biological liquid is placed on a non-porous and wettable solid substrate to dry, the “coffee ring effect” can be observed during drying. The enhanced evaporation flux at the

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pinned liquid-substrate contact line causes the outward capillary flow of liquid to replenish

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the evaporation loss at the contact line; this flow carries macromolecules, cells and other

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suspended particles to the edge of the drop, resulting in redistribution of these materials [10]. Because of the rapid loss of liquid and the consequential increase in the drop adhesion to the substrate, redistributed macromolecules deposit close to the contact line [11]. Further liquid

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evaporation may lead to the self-organisation of macromolecules, cells and other suspended particles on the solid substrate, forming patterns of well-defined morphologies when completely dried [12]. Among many dried drop patterns of biological liquid, the interesting patterns of the dried blood drop have been investigated by researchers for long time and attracted increasing attention. The history of scientific study on blood drop patterns started from the earliest publication by Piotrowski in 1895. He compared the blood patterns formed by mechanical impact on small animals in front of a white screen under different controlled conditions, and found that the appearance of blood patterns reflected the condition under which the mechanical impact was applied and also the source of blood [13]. In 1939, Balthazard 6

ACCEPTED MANUSCRIPT investigated the influence of the velocity of blood spatter to its pattern formation and indicated that the characteristic patterns could give decisive hints to the blood origin [14].

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Sixteen years later, Kirk recognized the importance of blood drop patterns in crime scene

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reconstruction and developed a related research project based on a famous murder case [13].

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In 1983, the International Association of Bloodstain Pattern Analysts (IABPA) was founded and the interpretation of blood drop patterns has become an effective tool in modern criminalistics laboratories. Since then, the evolution of blood pattern analysis in forensic

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science developed rapidly, and some theories have even been applied to the crime scene

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reconstruction, forensic education and training [15]. Actually, forensic science is mostly concerned with the blood drop impact rather than its deposition [16].

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Recently, many researchers are interested in the drying drop patterns of blood under static

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deposition. Such drying patterns are also known as desiccation patterns of blood drop. Several studies showed that variations of the concentration and chemical structure of

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macromolecules in blood can be identified from the morphological variations of dried drop patterns; this means that dried drop patterns of blood may carry important information of the

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health status of human and farming animals, and could be used for disease diagnosis [17-19]. In 2001, Kulyabina et al. used Wiener spectrum and Fourier transformation methods to analyse the crystal-like patterns of dried human plasma, and suggested that analysis on such patterns could be one of the most promising ways for diagnostics [20]. Rapis studied the pattern morphologies of dried plasma drop and indicated the potential use of these patterns for disease diagnosis in 2002 [21]. Subsequently, Yakhno et al. reported significant differences in patterns of dried blood serum among healthy people, pregnant women and patients suffering from different diseases, e.g., hepatitis, paraproteinemia, breast cancer, and lung cancer [22]. Most recently, Brutin et al. showed significant differences in the dried drop patterns of whole blood from healthy individuals and those from patients suffering from

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ACCEPTED MANUSCRIPT anaemia and hyperlipidaemia [18]. All of these studies demonstrated that a continuing research effort to the dried blood pattern analysis could make it an effective tool for simple

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and low-cost disease diagnosis.

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It is acknowledged that blood is a complex suspension of cellular components, e.g., mostly

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red blood cells (RBCs), white blood cells (WBCs), platelets, and a continuous plasma phase. The continuous plasma phase is composed of mostly water, proteins, inorganic electrolytes, and clotting factors [23, 24]. The cellular components and plasma play different roles in

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blood pattern formation. Accordingly, the dried drop patterns of plasma and whole blood will

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be reviewed separately.

2. Background of the drop evaporation

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2.1. The “coffee ring” effect

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When a drop of coffee is spilt on a solid substrate, a ring-shape deposit of coffee solids will

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form at the edge (contact line) of the drying drop. This interesting ring-shape deposit is the outcome of a physical phenomenon known as the “coffee ring” effect [25, 26]. The formation of the “coffee ring” is a ubiquitous phenomenon; it has been manifested in liquid systems

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with solid components of different sizes, ranging from macromolecular colloids to nanoparticles and individual molecules [27-30]. The “coffee ring” effect was first investigated by Deegan et al. [10]. These authors discovered the formation mechanism of the “coffee ring”. The enhanced evaporation rate at the pinned wetting contact line of the drop forces the liquid in the central part to flow outwards to replenish the faster liquid loss at the edge; this capillary flow could carry the suspended materials to the edge. After drop evaporation, these redistributed materials are concentrated at the drop edge, thus forming a “coffee ring”. There are three essential conditions for the ring formation: pinning of the liquid-substrate contact line, enhanced evaporation rate at the contact line, and suppression of Marangoni 8

ACCEPTED MANUSCRIPT flow [11, 31]. If the liquid-substrate contact line of the drop is not pinned, the radius of the drop will remain constant during the early stage of the drop evaporation. Meanwhile, the

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contact angle of the drying drop will decrease to a critical value. After that, the contact angle

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will remain constant, while the contact line of the drop will recede; the retraction of the

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contact line will drive the suspended materials move inwards. Therefore, this process could inhibit the ring formation. In contrast, if the contact line remains pinned and the liquid assumes as a wedge-like shape, the radius of the drying drop will remain constant. In this

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case, liquid at the contact line of the drop evaporates faster than that in the central part,

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resulting in the capillary flow from the centre to the contact line; this outward flow leads to the horizontal movement of solid components. Therefore, a “coffee ring” can be produced.

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However, for organic systems, e.g., the poly-methyl-methacrylate/octane and the mica

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flakes/octane, and for aqueous systems with special design of temperature field, the evaporation of the drop leads to the variation of the temperature, which influences the

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concentration of solid particles, generating the surface tension gradient along the drop-air interface; this surface tension gradient manifests as Marangoni convection within the drop

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[32-34]. In a drying drop, e.g., the organic system, the liquid on the surface of the droplet is pulled inward towards the top of the droplet, and then is plunged downwards, causing the circular movement along the drop-air interface. This circular liquid movement also carries the suspended solid components away from the regions with low surface tension to those of high surface tension, as shown in Fig. 1 [31]. As a consequence, the Marangoni flow could counteract the outward capillary flow, reversing the “coffee ring” phenomenon [35]. Apart from above three essential conditions, there are some other factors influencing the “coffee ring” formation. Yunker et al. experimentally demonstrated that the addition of ellipsoidal particles could suppress the outward transport of suspended particles to the contact line of the drop, and thus ensure the uniform deposition of particles. These researchers

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ACCEPTED MANUSCRIPT suggested that controlling the shape of suspended particles could be a convenient way to suppress the “coffee ring effect”, without any modification of the chemical properties of

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particles or solvent [36]. Our recent research indicated that the “coffee ring” might or might

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not be formed when the drop was placed on porous substrates, such as paper. This is due to

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the fact that the transport of suspended materials in paper is influenced by chromatographic and filtration effects. For some porous samples, in which the chromatographic and filtration effects are the dominant retarding forces to particle transport, the “coffee ring” could be

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inhibited [37].

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2.2. Current understanding of cracking patterns in colloidal drops Formation of cracking patterns is a commonly observed phenomenon in the drying

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colloidal drops on solid substrates. Cracking patterns with different morphologies have been

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reported in numerous literature articles; these patterns include wavy, straight, arched, spiral, circular, and three-armed cracks (Y-junction with 120° and T-junction with 90°) [38-44].

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A number of fundamental research works suggested that the non-uniform evaporation of a colloidal drop leads to the formation of three regions during drying: a dried region (solid

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state), a saturated region (gelled state), and a supersaturated region (liquid state) [45, 46]. The dried region starts at the contact line of the liquid drop and grows inwards as drying progresses. The saturated region, which is also known as the drying front, consists of consolidated solid particles that still remain wet [47]. Cracking usually starts at the drying front [46]. Dufresne et al. studied the drying process of liquid suspensions of monodisperse colloidal silica nanoparticles and observed that the cracking tips (the most front of the crack) in the saturated region were still remained wet [48]. These authors proposed that the growth of cracks could be related to the evaporation-induced capillary force on the drying film. Therefore, the drying behaviour of the colloidal drops are relevant to the formation of cracking patterns. 10

ACCEPTED MANUSCRIPT Apart from the redistribution of patterns in a drying drop, driven by the “coffee ring” effect, the continuous evaporation of liquid also promotes the particles to get closer. Further

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evaporation leads to the formation of tiny liquid menisci between particles on the top layer of

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the drop, which generates a negative Laplace pressure in the deposited film (compressive

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capillary force) [49-51]. The curvature of the liquid menisci increases as liquid evaporates, and thus the increase in the compressive capillary force between particles. This increasingly compressive capillary force leads to the water drainage from the particle clusters, and

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promotes the aggregation of the adjacent clusters, as shown in Fig. 2 [48, 50]. Subsequently,

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a particle film is formed on the top layer of the drop; this film will shrink during drying. However, the horizontal shrinkage of the saturated region of the drying drop is constrained by

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its adhesion to the substrate. The mismatch between the shrinkage and the substrate

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constraints results in the increasing tensile stress in the drying drop. Once the rising tensile stress exceeds the tensile strength at specific locations within the particle film, crack will

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occur first on the surface of the film to release the stress [52-55]. If the particles are hard, the film cracks easily [51]. Nevertheless, if the particles are soft, they can absorb more stress

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through deformation to close the pores on the film and also reduce the evaporation rate of the drop, delaying or even avoiding the cracking occurrence [51, 56]. Interestingly, most suspended particles in plasma and whole blood are soft. This leads to the fact that the drops of plasma and whole blood experience long drying time to form cracking patterns. We will first discuss in sequence of the drying processes of the plasma and whole blood drops.

3. Plasma patterns 3.1. Components in plasma Plasma is the liquid component of whole blood composed of mostly water (90% by mass), various proteins (6% by mass), inorganic electrolytes (1% by mass), glucose, and other minor components. Among these components, proteins and inorganic electrolytes are considered as 11

ACCEPTED MANUSCRIPT the main contributors to plasma pattern formation. There are three major types of proteins, i.e., serum albumin (45 g/L), globulin (25 g/L), and fibrinogen (3 g/L) in plasma of both

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human and domestic animals; the molecular weights of these three proteins are 65 kDa, 92

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kDa, and 340 kDa, respectively. The main components of inorganic electrolytes in blood

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plasma of a typical healthy human are Na+ (142 mmol/L) and Cl- (107 mmol/L) [57]. Moreover, the plasma is believed to be the Newtonian fluid [23].

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3.2. Morphologies of dried plasma drop

When a plasma drop is placed on a wettable solid substrate, protein macromolecules are

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driven towards the contact line by the “coffee ring” effect. As the liquid evaporates, gelation of plasma occurs initially at the edge of the drop due to the increase in the concentration of

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protein macromolecules. In this case, the inorganic electrolytes with high water solubility are

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driven by the receding liquid to the central part of the drop. Subsequently, further evaporation leads to the shrinkage of the gelled film of plasma drop, forming the cracking patterns after

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complete drying. On the other hand, the aggregated inorganic electrolytes in the central part of the plasma drop crystalize during drying, and produce the crystal patterns in this area.

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However, some researchers thought that protein macromolecules could also crystalize in the central part of drop [58-60]. This controversy will be further discussed in Section 3.5. The pattern morphologies of plasma drop from healthy human formed on the glass substrate under ambient condition are generally characterized by two zones, i.e., the central part and the peripheral region, as shown in Fig. 3. These regions are composed of two types of cracks, i.e., radial and orthoradial cracks, and various crystal-like patterns in the central part, such as dendrites, fractals, and inclusions [5, 61]. Bovine serum albumin (BSA) is a protein derived from the blood of cows, and has a wide range of applications in biomedicine. It is widely used as a blocking agent in enzyme-linked immunosorbent assay (ELISA); it also was used as a model protein to imitate human serum 12

ACCEPTED MANUSCRIPT albumin (HSA) for dried drop pattern research by Tarasevich et al. [62, 63]. The molecular weight of BSA is 66 kDa, and its solubility in water is larger than 40 g/L. The drying process

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of a BSA drop was classified into four main stages, including liquid volume loss, protein

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gelation, salt crystallization in semi-liquid gel, and thoroughly drying. The zonal structure of

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its dried drop patterns was then divided into four sections as shown in Fig. 4, i.e., (1) homogeneous protein film, (2) protein precipitates, (3) protein gel, and (4) crystallized salt area [64]. In further research, Chen and Mohamed used a fluorescence microscope to observe

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the central part of dried BSA drop patterns and found that crystals of several different shapes,

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such as rosette, scallop, Chinese arrow, and dendrite, were formed at ambient conditions [60].

3.3. Factors influencing plasma pattern formation

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3.3.1. Influence of inorganic salts

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Inorganic salts play a critically important role in the formation of crystal-like patterns in dried biological materials. Annarelli et al. investigated the influences of various bio-

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medically important metal chlorides, such as LiCl, NaCl, KCl, CaCl2, MgCl2, and FeCl3, on the morphologies of dried drop patterns of BSA solution and observed different structures.

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These authors suggested that the differences in structure could be related to the properties of the cations of salts dissolving in the drop [65]. Yakhno et al. further reported that the crystallike patterns could not form in a drying drop of BSA solution without salt [66]. There have also been several experimental works on the influence of salt concentrations on the morphologies of the dried drop patterns of BSA solution. Yakhno et al. suggested that dried drop patterns of BSA solution were possibly related to the NaCl-induced transition of protein phase from solution to gel (gelation process) during drying [64]. Furthermore, Chen and Mohamed observed two different crystallization processes of the inorganic salts in the drying drops of BSA in the PBS (phosphate buffer solution) with different salt concentrations: the salt crystals first appeared at the edge of the drop of low salt concentration (8 g/L), but 13

ACCEPTED MANUSCRIPT initially occurred at the central part of the drop of high salt concentration (80 g/L). Thus, they proposed two different evaporation modes for the drying drop of BSA/PBS, i.e., the enhanced

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evaporation rate at the edge of the drop for the low salt concentration solution and the higher

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evaporation rate in the centre for the high salt concentration solution [60].

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On the other hand, the pattern morphologies of human serum albumin (HSA) are also influenced by inorganic salts significantly. Buzoverya et al. investigated the impact of NaCl on the morphologies of dried drop patterns of HSA [67]. They compared dried drop patterns

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of a series HSA from 0.2 to 10 g/L in water and in 0.9 g/L NaCl saline solution. They found

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that the dried drop patterns obtained from water solutions had fewer visible structures than those from saline solution. They suggested that NaCl could promote the dehydration of the

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protein macromolecules (HSA) and allow stronger intermolecular interaction among HSA,

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favouring the aggregation of HSA and the subsequent gelation process. This could be due to the thinning of electric double layer around the HSA upon increasing the NaCl concentration

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(further discussion of applying the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory to the interpretation of drying drop patterns will be presented in Section 3.4).

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3.3.2. Influence of the protein concentration The morphologies of the dried patterns of plasma were reported to be strongly dependent on the protein concentration [67]. For better understanding of the relationship, BSA and HSA were studied, respectively. Chen and Mohamed observed the star-shaped patterns in the dried drop of low concentration of BSA (0.01 mg/mL) in PBS, while the spear-head-shaped patterns in 0.05 mg/mL of BSA in PBS. When the BSA concentration reached 0.1 mg/mL, the rosette patterns began to form and their sizes increased with the increase in the protein concentration [60]. Tarasevich et al. investigated the patterns obtained from more concentrated BSA in saline solutions. They observed radial cracks generated in the dried drop patterns of all BSA

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ACCEPTED MANUSCRIPT concentrations. They further found the dendritic patterns with long straight and short curved branches in the dried drops of BSA/saline solutions of 80 g/L and 90 g/L; these patterns

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changed to chains of fine crystalline structure in 100 g/L BSA/saline solution. After further

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increasing the BSA concentration to above 110 g/L, the dendrites appeared again with

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flower-shaped patterns appearing in its central part of the dried drop [62]. Buzoverya et al. studied the influence of the protein on the dried drop patterns through studying the evolution of the morphologies of the crystal-like patterns driven by the

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increasing concentration of HSA in saline solution. As the HSA concentration increased from

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0.2% to 10% g/L, the morphologies (shown in Fig. 5) transformed from (a) layered porous micelles to (b) dendrites, and to (c) fractal, and then to (d) spheroidic structures, and finally

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to (e) a glassy matrix with uniformly arranged spherical inclusions [67]. Detailed

discussed in this study.

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physicochemical analysis of the evolution of the dried drop patterns was, however, not

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3.3.3. Influence of the plasma concentration and substrates Karen et al. diluted the freeze-dried human plasma with purified water by volume to 10%

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(i.e., water : plasma = 90% : 10%), 25%, and 50%, and then deposited drops of these plasma solutions onto controlled substrates with different final apparent “contact angle” (θ): fused silica (θ ≈ 32°), gold-coated glass (θ ≈ 53°), calcium fluoride (θ ≈ 71°), and Teflon coated stainless steel (θ ≈ 89°) (SpectRIM) [68]. These authors observed that the dried plasma drops consisted of the smooth film-like ring in the edge and the crystal-like patterns in the central part, as shown in Fig. 6. However, the length of radial cracks of the dried plasma drop decreased with the decrease in the plasma concentration. Since the dilution of plasma changes the amount of liquid and the rheology of the plasma drop, e.g., viscosity, the evaporation process could be changed. As a result, the length of radial cracks differs from the drop with different plasma concentrations. Nevertheless, the detailed explanation is still insufficient.

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ACCEPTED MANUSCRIPT Among the dried plasma patterns illustrated in Fig. 6, they also found that significant changes in the pattern morphologies of the dried plasma on different substrates; their

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observations suggested that the dried drop patterns of plasma were related to the wettability

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of substrates. In order to further investigate the influence of the substrates on the dried plasma

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drop, they employed Raman spectroscopy with the excitation wavelength of 785 nm to analyse the chemical composition of the proteins in the edge. Their experimental results indicated that the substrates had a negligible influence on the chemical compositions and

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molecular structures of the proteins in the edge of the dried plasma drop [68].

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3.4. Driving forces of cracking

When a drop of human plasma is placed on a solid substrate and allowed to dry, the radial

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capillary force due to the “coffee ring” effect drives protein macromolecules radially

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outwards; this increases the protein concentration near the contact line. With the continuous evaporation, some protein macromolecules will deposit on the edge of drop, whilst protein

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macromolecules remaining in solution are exposed to a solution of higher salt concentration and therefore also higher ionic strength. According to the DLVO theory, an increase in ionic

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strength suppresses the thickness of the electric double layer around the protein macromolecules, thus weakening the electrostatic repulsion among protein macromolecules. This situation favours the aggregation of protein macromolecules [60]. As the evaporation of liquid progresses, aggregated protein macromolecules experience a gelation process, which begins to form a film on the dried region of the drop. In the initial stage of gelation, the edge of drop solidifies, while the central part still remains as liquid [69]. The evaporation-induced shrinkage starts from the edge of the plasma drop. Subsequently, owing to the competition between the film shrinkage and the adhesion of the gelled protein macromolecules to the substrate, stress in this film builds up. Cracking on the film develops as a way to dissipate the stress in the film. 16

ACCEPTED MANUSCRIPT Interestingly, two types of cracking patterns are formed during further drying. Annarelli et al. observed that the radial cracks occurred first, followed by the orthoradial cracks. They

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suggested that the initial radial cracks were caused by the competition between the

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evaporation-induced orthoradial shrinkage and the adhesion of gelled proteins to the substrate.

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In the vicinity of a radial crack, the shrinkage stress was clearly parallel to the edge. Once the radial crack stopped, the cracking was observed to sequentially turn to the orthoradial direction [70]. However, the fundamental reasons of this observation were not given. More

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recent researches indicated that the radial cracks in a drying colloidal drop could be related to

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the remaining liquid in its central part during gelling [49, 71, 72]. The gelation of protein macromolecules starts from the contact line of the drop, while the central part is still liquid.

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The surface tension of the hemispheric cap of the liquid drop in the central part may cause the

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radial stress on the gelled zone, leading to the formation of flaws in the radial direction. Subsequently, further evaporation of liquid results in an increase in the shrinkage force acting

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on the gelled zone, and thus produces the radial cracks. The radial cracks propagate from the edge of the drop to the central part [49]. However, there is no direct experimental result

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supporting this hypothesis for the radial cracks in the dried plasma drop. Hence, the fundamental mechanisms of cracking formation of dried plasma patterns are still unclear.

3.5. Mechanisms of the central pattern formation Several groups have published their experimental works to interpret the underlying mechanisms of the formation of crystal-like patterns in the central part of the dried plasma drop [58-60, 73, 74]. However, controversies over the formation of crystal-like patterns in the central part of the dried plasma drop still remain. Some researchers claimed that the crystal-like patterns were made up of protein macromolecules and inorganic salts. Chen and Mohamed used the fluorescent microscope with an excitation wavelength of 488 nm to observe the fluorochrome labelled albumin, 17

ACCEPTED MANUSCRIPT which has an emission wavelength of 520 nm; their results suggested that the crystal-like patterns were albumin [60]. After that, other researchers experimentally studied protein

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crystallization and employed the DLVO theory to explain this phenomenon of the crystal

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pattern formation. Their reasoning was that the compression of the electric double layer

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around protein macromolecules by the increasing concentration of salts would lead to the aggregation of the macromolecular proteins and the formation of a gelled matrix in areas except the central part of the drying drop. The concentrated salts in the central part crystallize

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during further drying. The dried patterns in the central part of plasma drop were thus thought

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to be composed of aggregation of proteins and the crystals of salts [58, 59]. However, Yakhno proposed that the protein macromolecules could only form the gelled

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matrix; the observed crystal-like patterns were crystalized salts on the gelled protein matrix.

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This opinion is different from Chen and Mohamed‟s experimental results [73]. In order to support his hypothesis, Yakhno carried out the experiments based on the re-dissolution

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method. He first stuck the dried drop patterns of BSA/NaCl to the glass substrate by heating, and then rinsed it with distilled water; he observed that the crystal-like patterns disappeared

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completely. In the second experiment, he immersed the dried drop patterns into a 70% ethanol solution for 30 min followed by drying at room temperature, and then found that the crystal-like patterns still remained on the glass substrate. Yakhno suggested that the NaCl crystals could not completely dissolve in the ethanol. Thus, he thought that the remaining patterns could be salt crystals [74]. These results provided circumstantial evidence supporting his opinion that the crystal-like patterns were mainly of the salts with good water solubility. Based on these results, they suggested that the complex crystal-like patterns in the dried drop of BSA/NaCl solution consisted of only salt (NaCl) crystals, with proteins only serving as seeds for their growth [74].

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ACCEPTED MANUSCRIPT Actually, it is a difficult task to experimentally distinguish the protein crystals from those of salts in dried drop patterns of plasma without special and advanced analytical techniques.

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Therefore, more research is needed to gain further understanding of the nature of the

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crystalline patterns in the central part of the dried plasma drop and their formation

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mechanisms.

4. Whole blood patterns

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4.1. Components in whole blood

Whole human blood contains plasma (55% by volume) with macromolecular proteins and

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inorganic salts, as well as three groups of cellular components (45% by volume), i.e., red blood cells (RBCs), white blood cells (WBCs), and platelets. RBCs, WBCs, and platelets

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represent 97%, 2%, 1% of the total volume of these cellular components, respectively [18].

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RBCs, in the blood from healthy people, are the donut-shaped and soft colloidal particles of 8

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µm in diameter by 2 µm in thickness. The concentration of RBCs (or haematocrit) can influence the physical properties of whole blood, such as the viscosity [16]. Furthermore, Sobac and Brutin investigated the rheology of whole human blood and suggested that blood

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behaves like a non-Newtonian fluid [75]. Therefore, the morphologies of dried drop patterns of whole blood are significantly different from those of plasma.

4.2. Morphologies of dried blood drop The morphologies of dried drop patterns of blood from healthy human formed on the glass substrates are composed of three obvious zones: a central part with small and chaotic cracks (small sized plaques), a coronal region with large cracks (large sized plaques), and a fine peripheral area, which adheres to the substrate (shown in Fig. 7).

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ACCEPTED MANUSCRIPT 4.3. Process of cracking pattern formation Drying process of the blood drop on clean microscope glass slides were investigated by

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Brutin et al., as shown in Fig. 8. These authors showed that the drying blood drop

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experienced five stages of morphological changes. These stages were: pre-gelation (stage 1),

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gelation (stage 2), cracking in the peripheral area and coronal region (stage 3), completion of gelation (stage 4) and formation of the small-scaled disordered cracks in the central part

NU

(stage 5) [18, 75].

Moreover, Brutin et al. suggested that the drying process of the blood drop was limited by

MA

the diffusion of liquid into the air. Hence, they used the Stephan‟s diffusion model (equation 1) to calculate the total time of liquid evaporation (t) of a blood drop, and fitted this model

D

with their experimental observations. Stephan‟s diffusion model is applied for modelling the

TE

evaporation from a flat and circular liquid surface [76]. This model suggests that the evaporation rate of the liquid is proportional to the radius of the drying liquid surface, as well

(1)

AC

air [77-79].

CE P

as the difference between the saturated vapour pressure of the drying liquid surface and the

where R is the gas constant, T is the air temperature, m0 is the initial mass of blood drop,

P

is the pressure difference between the saturated vapour of the water in blood drop and the air, d0 is the initial wetting diameter, and Ddiff is the diffusion coefficient of the water into air.

4.4. Factors influencing on blood pattern formation 4.4.1. Influence of the relative humidity Zeid and Brutin noted that the size of plaques in the coronal region increased as the relative humidity (RH) increased from 13.5% to 50.0%, but decreased with the further increase of RH

20

ACCEPTED MANUSCRIPT from 50.0% to 78.0%. By contrast, the width of the peripheral area increased continuously, which is indicated by the yellow label in Fig. 9 [80].

T

In a further study, Zeid and Brutin measured the spreading rate of blood drops on

IP

microscope glass slides under different RH values ranging from 8.0% to 90.0% at the same

SC R

temperature [81]. Their results showed that blood drops spread more rapidly on glass under the low RH than the high RH [81]. These authors observed that the final apparent “contact angle” (θ) of blood drop on the glass surface decreased linearly with the increase of the RH

(2)

MA

NU

from 13.5% to 78.0%, and then proposed a fitting equation as follow: [80].

where the RH is the controlled relative humidity ranging from 13.5% to 78%.

D

Based on these observations, Zeid and Brutin suggested that the evaporation rate of whole

TE

blood drop on the glass substrate is dependent on the relative humidity of the environment. Hu and Larson experimentally investigated the evaporation of a sessile drop by using the

CE P

finite element method and derived a simple approximate evaporation rate expression for the drop with the final apparent “contact angle” less than 90° [82]. Since the contact angle of

AC

blood drop on the glass surface is below 40° (equation 2), Zeid and Brutin employed the Hu and Larson‟s model to calculate the evaporation rate (E) of a blood drop: (3)

where Cv is the water vapour concentration at the top of the centre (apex) of blood drop [80]. Accordingly, the higher RH leads to the lower evaporation rate, which may prolong the drying time of the blood drop. The liquid will be remained in the blood drop with longer time; this could influence on the adhesion of the drop to the glass substrate. Consequently, the size of plaques could be different in the coronal region of the dried blood drop formed under different RH [79]. On the other hand, the higher RH results in the smaller final apparent “contact angle” of the blood drop; this also could lead to the smaller height and the larger 21

ACCEPTED MANUSCRIPT width of the pinned contact line (wedge). As a result, the width of the peripheral area of the dried blood drop increases as the RH. However, it is still unclear as to whether and how the

T

changed evaporation rate influence the materials transport within the drop, and consequently

IP

the gel transition in the dried film.

SC R

4.4.2. Influence of substrates

Brutin et al. investigated the difference between the role of the thermal property of substrates and the role of the wettability of substrates in influencing the evaporation

NU

dynamics of blood drops [16]. These authors observed the drying blood drops on different

MA

metallic substrates with the similar drop wettability, i.e., gold and aluminium. After comparing the heat and mass transfer of blood drops on these substrates during drying, they

D

indicated that the thermal diffusivity of substrates has insignificant influence on blood

TE

evaporation dynamics [16]. At the same time, they found a non-uniformed solid skin on the surface of gelling blood drop on glass with the final apparent “contact angle” (θ) of 20.5°. In

CE P

contrast to the glass surface, a uniform glassy skin formed on the surface of blood drop on the metallic substrates, i.e., gold and aluminium, with the final apparent “contact angles” of

AC

above 90°, as shown in Fig. 10 [16]. Such a drying patterns difference was attributed by the authors to the fact that the evaporation flux from the peripheral region of a low contact angle drop on a substrate (θ < 40°) is higher than that from the central part of the drop. However, for a blood drop deposited on a non-wetting surface (θ > 90°), the difference in evaporation flux of the peripheral region and the central part of the drop would be small [16, 82]. Drying of blood drop on the non-wetting substrates would therefore yield different patterns from those on the wetting substrates [16]. On the other hand, the final wetting radius of the blood drop on the glass substrate is different from that on the metallic substrates. Assuming that the shape of the blood drop on the solid substrate is the hemispheric cap, the drop volume (V) can be calculated as following:

22

ACCEPTED MANUSCRIPT (4) where R is the final wetting radius of the blood drop on the solid substrate [83]. Accordingly,

T

for a given volume of the blood drop, the final wetting radius of that on the glass substrate (θ

SC R

4.4.3. Influence of the blood drop diameter and height

IP

= 20.5°) is larger than that on the gold (θ = 91.9°) and aluminium (θ = 95.7°).

Brutin et al. found that the number of cracks in the coronal region of a dried blood drop

NU

increased steadily with the increase in diameter of the three phase contact line of the blood drop on a wettable substrate [18]. These cracks were wide and axisymmetric about the centre

MA

of the drop. For drops that have contact line diameters smaller than 4.2 mm, no obvious cracks could be observed in the coronal region. However, as the contact line diameter

D

increased to larger than 5.4 mm, cracks appeared in the coronal region at periodic spacing

TE

[18]. This observation was related to the stress developed inside the blood drop during drying. For small drops with small diameter of contact lines (< 4.2 mm), the stress is much lower

CE P

than that in the drops of the large diameter of contact lines. In addition, the initial crack spacing (λ) was roughly proportional to the maximum height

AC

of gelled blood drop (h), according to the model proposed by Allian and Limat [75]: (5)

where J0 is the flux of water evaporation, Dm is the diffusion coefficient of water transport in the porous gelled matrix, and C∞ is the volume fraction of the evaporated water. The rationale of this empirical relationship was not further discussed by the authors, but the Allian and Limat model (equation 5) appeared to be a more accurate prediction of the experimental data on blood drop collected by Sobac and Brutin [75].

23

ACCEPTED MANUSCRIPT 4.5. Mechanisms of pattern formation Comparing with plasma, the major suspended “soft solids” in whole blood are cells of

T

several micrometres in size, mostly RBCs. Hence, RBCs play an important role in pattern

IP

formation of whole blood.

SC R

When a blood drop is placed on the glass substrate, due to the non-uniformed evaporation rate of the blood drop, a “coffee ring” of RBCs occurs at the pinned contact line. Evaporation

NU

leads to the decrease in the height of the liquid film at the contact line, thus separating the suspended colloid particles in whole blood drop according to their sizes. The smallest

MA

particles are in the forefront of the contact line; large particles are sequentially behind the small ones. In a drop of whole blood, RBCs cannot be pushed to the very edge of the drying

D

drop. Instead, the macromolecular proteins of smaller sizes can reach the edge of the pinned

TE

contact line. Thus the suspended colloidal particles in whole blood drop on the glass substrate could be classified at the contact line according to their sizes [75, 81, 84].

CE P

Sobac and Brutin suggested that the competition between the adhesion of blood drop to the glass substrate and the evaporation-induced shrinkage of the drop led to the formation of

AC

cracks [75, 80]. These authors proposed that continual evaporation of liquid could lead to the formation of the plasma meniscus between the cells at the drop-air interface, generating the Laplace pressure to pull the cells towards one another. The curvature of meniscus increased with further evaporation, leading to the increase in the magnitude of the Laplace pressure; this could generate the increasing compressive forces between cells and unite them together. Since the aggregated cells are soft and elastic matters, they could be deformed after suffering from the compressive forces. Therefore, a small-sized porous network with entrapped fluid could be formed on the top layer of the drying blood drop; this network could prevent the evaporation of the blood drop. On the other hand, a depletion model was recently proposed to explain the aggregation of RBCs in solution [85, 86]. During drying, the RBCs inside the 24

ACCEPTED MANUSCRIPT blood drop get gradually closer to one another. Once the distance between RBCs is less than the size of protein macromolecules in blood, these protein macromolecules between RBCs

T

will be expelled out of the regions between RBCs. As a result, the region between RBCs will

IP

experience a depletion of macromolecules, thus producing an osmotic pressure between

SC R

RBCs. The osmotic pressure could be the main contributor to the aggregation of RBCs within the blood drop. Further aggregation could lead to the formation of the large-sized plaques. Recently, Xu et al. employed the Johnson-Kendall-Roberts (JKR) model to calculate the

NU

adhesion force between RBCs to simulate the aggregation of the RBCs [87]. JKR model is a

MA

model describing the strong adhesion energies of soft and deformable materials [88]. They suggested that the strong intermolecular adhesion among elastic particles caused the

D

formation of the „neck‟ around the contact area due to the infinite stress at this area [88]. Chu

TE

et al. used a micropipette technique to measure the force necessary to separate the adhering cells and showed that the cytoskeleton provides the cells with a three-dimensional structure

CE P

with good elasticity; this structure enabled the slight deformation of the adhering cells. This mechanism is in agreement with the JKR model [88]. Accordingly, the aggregation of RBCs

AC

could occur with the increasing number of the adhering RBCs [87]. Gelation of blood drop happens with the increasing aggregation of RBCs during drying. The gelled blood drop shrinks inwards as the water evaporates. However, the shrinkage of the gelled blood drop is limited by its adhesion to the substrate, resulting in the build-up of tensile stresses on the drying blood drop. For releasing the increasing tensile stress within the gelled blood drop, cracks first appear at the contact line and extend towards the centre of the blood drop [75, 80]. The time for the initial cracks to form in the peripheral region of blood drop was reported to be about 25% of the total drying time, and that of cracks in the central part formed at approximately 44% of the total drying time [89]. As the liquid completely evaporates, the whole cracking patterns are formed.

25

ACCEPTED MANUSCRIPT During drying, the RBCs accumulate in the coronal region of blood drop, and then produce the large sized plaques containing many RBCs after drying. Nevertheless, the central part of

T

blood drop contains mainly plasma proteins and forms small-sized and chaotic plaques [18].

IP

Sobac and Brutin suggested that different patterns of the coronal region and the central part

SC R

of dried blood drop could also be related to different gelation processes, as shown in Fig. 8. These authors observed that the initial gelation occurred at positions between the peripheral region and the coronal region of blood drop; then the gelling front propagated inwards.

NU

Afterwards, the gelation of the central part occurred quasi-simultaneously [75]. However, no

MA

further explanations to the gelation process were reported. Interestingly, the circular patterns with bright red colour are formed on most of the large-

D

sized plaques in dried blood drop, surrounding by the patterns with dark red colour, as shown

TE

in Fig. 9. Sobac and Brutin attributed this observation to the adhesion of the central part of dried blood plaques to the glass substrate and the delamination of their edges from the

CE P

substrate, respectively [75]. After the formation of the larger-sized plaques by cracking in the coronal region, the liquid continues to evaporate. The drying surface on the upper layer (air-

AC

drop interface) of the plaques suffers from more shrinkage than the under layer (dropsubstrate interface), which is still wet. At the same time, the compressive force on the drying surface of the large-sized plaques increases gradually. This compressive force could lift the edges of the plaques and generate the out-of-plane deformations of the large-sized plaques. Therefore, the circular patterns with bright red colour are thought to be the areas of final adhesion between the dried blood drop and the substrate, while the rest part with dark red colour indicates the detachment of the blood pattern from the substrate [39, 75, 90]. Nevertheless, no further and convinced experimental evidences are supported for this hypothesis.

26

ACCEPTED MANUSCRIPT 5. Applications 5.1. Medical diagnosis

T

Interpretation of morphologies of dried human blood patterns could potentially be used as

IP

an effective tool to evaluate human health status, since the patterns can reflect the variation of

SC R

blood compositions caused by disease.

The applications of dried blood patterns on medical diagnosis were primarily carried out

NU

from plasma. Rapis was the first researcher to use the dried human plasma patterns to diagnose metastatic carcinoma [21]. Subsequently, Buzoverya et al. found that various

MA

interesting patterns of dried human plasma drop could be correlated to diseases. These authors then suggested that different cracking patterns of dried human plasma (Fig. 11) could

D

reveal different pathological information [61]. For instance, the triactinal cracks (Fig. 11 (a))

TE

may indicate disorders in the venous outflow from the cranial cavity and the congestive

CE P

events (including those in cerebral tissues). The tourniquet shape cracks (Fig. 11 (b)) are related to the chronic hypoxia and the inflammation. The dished cracks (Fig. 11 (c)) can be the forecast for the early stage of encephalopathy. The large sized cracks (Fig. 11 (d)) provide

AC

the pre-diagnosis of the disorders in water balance of the body (dehydration and dysproteinemia). Yakhno et al. compared the dried drop patterns of plasma from healthy people and patients suffering from viral hepatitis B and burn-related disease, and found that the drying times of plasma drops from patients were longer than those from healthy people [91]. More recently, Muravlyova et al. observed the significant differences between dried drop patterns of plasma from patients with interstitial lung fibrosis (ILF) and those from the patients with idiopathic interstitial pneumonia (IIP); they suggested that the differences in dried plasma patterns could be linked to the modified proteins and extracellular nucleic acids caused by the interstitial pulmonary disease [92].

27

ACCEPTED MANUSCRIPT Apart from the plasma, the dried drop patterns of blood serum were also suggested to be useful for disease diagnosis. Blood serum is the plasma without fibrinogens; it contains all

T

proteins that have no function for blood clotting (e.g., serum albumin and globulin) and all

IP

the electrolytes, antibodies, antigens. Because plasma has similar biological components to

SC R

serum, their dried patterns formed under the same condition are similar. The camomile shape patterns with equal intervals of radial cracks were typically found in the serum of healthy individuals. In contrast to this, more chaotic cracks could be observed in serum patterns from

NU

patients under chronic health conditions. The authors, however, did not provide details about

MA

patients‟ health conditions [93]. Later studies by Yakhno et al. presented systematic correlations and analyses of specific cracking features in dried serum patterns from patients

D

suffering from breast cancer, lung cancer, hepatitis, and paraproteinemia, as well as from

TE

women with premature deliveries in different periods (Fig. 12). These authors further measured the blood serum samples using the Acoustical-Mechanical Impedance (AMI)

CE P

method and showed that the dried drop patterns of blood serum samples from healthy individuals could be easily distinguishable from those obtained from patients suffering from

AC

above mentioned diseases [17, 22]. AMI is a device containing a drop-loaded crystal resonator and an amplitude detector; this device can be connected with personal computers and can detect the physical properties of the dying drops, i.e., weight, density, viscosity, elasticity, and the friction coefficient and adhesion strength of the drop to the substrate [94, 95]. Shabalin and Shatokhina investigated on the blood serum patterns of the patients with inflammation, sclerosis, and chronic intoxication, and showed that some characteristic morphologies of blood serum patterns could be used to acquire information about the health state of human organism [96]. There have been several publications devoted to correlating the pattern morphologies of dried drop of plasma or serum with human health conditions, however, the use of whole

28

ACCEPTED MANUSCRIPT blood patterns for medical diagnosis was rarely reported [16]. In order to study patterns of plasma and serum drops, separation of plasma and serum from whole blood must be

T

performed first. This requires extra sample preparation steps. Moreover, the plasma and

IP

serum samples could be contaminated by haemolysis of RBCs [97, 98]. As a consequence,

SC R

some researchers investigated whole blood patterns for clues to visually and directly correlate the patterns of dried whole blood drop with human health conditions. Brutin et al. studied the dried drop patterns of whole blood from sick and healthy individuals (Fig. 13 (a) and (c)), and

NU

found that the peripheral region and the outer area of the coronal region of dried blood drop

MA

were light-coloured and had more intermediate-sized plaques for individuals suffering from amaemia (Fig. 13 (b)), whilst for individuals with hyperlipidaemia, these areas were greasy

D

(Fig. 13 (d)).For these unhealthy conditions, the dried blood drop patterns do not have radial

TE

cracks in the coronal region, instead, they show a larger number of smaller sized plaques in the central region [18]. By interpreting these observations, the light-coloured peripheral area

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and outer coronal area of patterns of dried blood drops were most likely to be associated with the smaller number of RBCs and the low level of haemoglobin in RBCs of anaemia sufferers.

AC

The high grease content was most likely to be related to the partial phase separation of blood lipids and plasma during drying.

5.2. Animal husbandry detection Recently, the dried drop patterns of plasma and whole blood from diseased cows with leukaemia and tuberculosis were reported to have specific morphological features that were significantly different from their healthy counterparts. In order to quantitatively distinguish the dried drop patterns of plasma and blood of healthy cows from those suffering from these diseases, the properties of the drops, such as drop mass, friction of the drying drop on the substrate, adhesion of the drop to the substrate and the drop viscoelasticity, were monitored by AMI during the drops drying process. As a result, significantly different AMI results were 29

ACCEPTED MANUSCRIPT obtained from the drying drops of healthy and diseased cows [99]. This has been the first publication on the application of dried drop patterns of plasma and blood in animal health; it

T

opens a possible new research area of simple disease detection in animal husbandry.

IP

5.3. Limitations of applications

SC R

Although the medical diagnosis from blood patterns has been demonstrated to be low-cost and fast, it has some limitations as a clinical test. Firstly, pattern morphologies of the dried

NU

drop of blood or plasma are sensitive to the drying condition (e.g., substrate and RH); successful tests would then require environment with controlled temperature and relative

MA

humidity. Secondly, individual diet could contribute to the variation of blood compositions and RBCs concentration, and thus changes the pattern morphologies of dried blood drop [12].

D

Thirdly, the diagnosis is subjective, since it relies on the visual perceptions of researchers.

TE

Despite these limitations, blood drying patterns contain much information which can be extracted and evaluated in future by image analysis and computer modelling.

CE P

6. The future perspectives

Research on the blood drop pattern-based medical diagnosis is still in a very early stage.

AC

Significant research efforts will be needed to nurture this new field of research to the stage of medical applications for both human and farming animals. In the current stage, the fundamental principles of blood pattern formation are not fully understood; this hinders the development of its applications. The gelation process of drying blood drop is still not clear. In addition, our understanding on factors influencing the morphologies of blood drop patterns is insufficient; this may mislead researchers to draw incorrect conclusions from their observations. Therefore, further research on the mechanistic understanding of blood pattern formation and biophysical interaction in the cellular and molecular levels should be the main foci of this research field.

30

ACCEPTED MANUSCRIPT The main components in blood and its physical and biological properties, e.g., viscosity, viscoelasticity, and clotting capability, can be changed by some diseases; these variations can

T

significantly change the pattern morphologies of dried blood drop. Hence, the linkage

IP

between diseases and dried blood patterns should be further studied.

SC R

The dried drop patterns of blood can be influenced by the environmental conditions. In order to better control the accuracy of the morphological analysis on the dried blood patterns, the influence of environmental conditions on the dried drop patterns of plasma and whole

NU

blood will be investigated.

MA

Objective assessment system will need to be established to remove subjective evaluation. Although some new technologies have been designed to overcome human subjectivity, such

TE

need to be developed [99, 100].

D

as the Acoustical-Mechanical Impedance (AMI), more stable and accurate technologies will

In conclusion, blood pattern-based medical diagnosis, as a new and rapid method for

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disease detection, will need more time to embody its real value to the society.

Acknowledgements

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Authors thank Miss Hui He from the Fashion and Art Design Institute, Donghua University, Shanghai, China, for her kind help in drawing of diagrams. Ruoyang Chen thanks Monash University Institute of Graduate Research and the Faculty of Engineering for postgraduate research scholarships. Duyang Zang thanks the National Natural Science Foundation of China (Grant No. 51301139). Wei Shen and Liyuan Zhang gratefully acknowledge Australian Research Council Discovery Project (ARC DP1094179).

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ACCEPTED MANUSCRIPT Figure captions Figure 1 Flow field in a drying octane droplet containing fluorescent poly-methyl-

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methacrylate particles, (a) experimental image and (b) theoritical prediction by Hu and

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Larson. The red arrows represent the direction of circular flow caused by the Marangoni

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effect [31].

Figure 2 A schematic diagram of the liquid menisci in a drying colloidal drop; the concave

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menisci indicate the negative pressure inside the drying droplet. The red spheres represent the suspended particles; the green arrows represent the compressive forces between particles.

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Figure 3 Morphologies of dried drop patterns of plasma from a healthy individual [5, 61]. Figure 4 Zones in the dried drop patterns of BSA solution: (1) homogeneous protein film; (2)

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protein precipitates; (3) protein gel; (4) crystallized salt area [64].

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Figure 5 Major morphologies observed in the central part of the dried drop patterns of HSA/saline solution of different HSA concentrations: (a) 0.2-0.6% g/L; (b) 1.0-2.0% g/L; (c)

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4.0-6.0% g/L; (d) 8.0-9.5% g/L; (e) 10.0% g/L [67]. Figure 6 Drops of human plasma dried on various substrates at different concentrations: (a)

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undiluted plasma on the SpectRIM; (b) 50% of plasma on the SpectRIM; (c) 25% of plasma on the SpectRIM; (d) undiluted plasma on the calcium fluoride; (e) 50% plasma on the calcium fluoride; (f) 25% plasma on the calcium fluoride; (g) undiluted plasma on the gold; (h) 50% plasma on the gold; (i) 25% plasma on the gold; (j) undiluted plasma on the fused silica; (k) 50% plasma on the fused silica; (l) 25% plasma on the fused silica [68]. Figure 7 Dried drop patterns of whole blood from a healthy individual on a microscope glass slide at room temperature (22 °C); relative humidity was not specified [18]. Figure 8 Drying process of whole blood from a healthy individual formed on a microscope glass slide at 22°C (100 s between frames) [18].

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ACCEPTED MANUSCRIPT Figure 9 Morphologies of whole blood drops formed on microscope glass slides under various RH values at the range between 13.5% and 78% [80].

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Figure 10 Morphologies of dried blood drops formed on different substrates: (a) glass; (b)

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gold; (c) aluminium [16].

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Figure 11 Different cracks in plasma patterns of people with various health conditions: (a) triactinal crack; (b) tourniquet shape crack; (c) dished crack; (d) large-sized crack [61] (read text for details).

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Figure 12 The morphological features of dried drop patterns of blood serum from (a) healthy

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individuals; patients suffering from different diseases: (b) breast cancer; (c) lung cancer; (d) paraproteinemia; (e) maternal full term delivery; (f) maternal premature delivery; (g)

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threatened abortion (premature delivery) in different periods of gestation; (h) hepatitis [22].

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Figure 13 Pattern morphologies of whole blood from (a) 27-year-old healthy woman; (b)

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patient with anaemia; (c) 31-year-old healthy man; (d) patient with hyperlipidaemia [18].

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Graphical Abstract

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ACCEPTED MANUSCRIPT Highlights 1. Drying process of sessile drops

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2. The formation of dried drop patterns of plasma and whole blood

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3. Potential applications of dried drop patterns of plasma and whole blood

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