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Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"6989",leadTitle:null,fullTitle:"Biogenic Amines in Neurotransmission and Human Disease",title:"Biogenic Amines in Neurotransmission and Human Disease",subtitle:null,reviewType:"peer-reviewed",abstract:"Drawing on the expertise of experienced researchers in neurotransmission and catecholamines, this book provides a brief overview of the latest knowledge in the field. 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The book is imagined as a comprehensive approach that will include all related topics and detailed information starting from production to consumption, composition of herbs and spices, and their contribution and mechanisms for treating various ailments, helping readers in making correct food choices for betterment of health and contribution towards a healthy society.
",isbn:"978-1-83969-609-1",printIsbn:"978-1-83969-608-4",pdfIsbn:"978-1-83969-610-7",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"f95ecdf9c56db9567aa29b880dba5836",bookSignature:"Dr. Rabia Shabir Ahmad",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10907.jpg",keywords:"Herb, Spice, Functional Food, Essential Oil, Antioxidant, Health Benefit, Garlic, Cinnamon, Black Cumin, Tamarind, Curcumin, Clove",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 1st 2021",dateEndSecondStepPublish:"March 29th 2021",dateEndThirdStepPublish:"May 28th 2021",dateEndFourthStepPublish:"August 16th 2021",dateEndFifthStepPublish:"October 15th 2021",remainingDaysToSecondStep:"a month",secondStepPassed:!1,currentStepOfPublishingProcess:2,editedByType:null,kuFlag:!1,biosketch:"Dr. Rabia Shabir Ahmad won various research projects funded by Higher Education Commission (HEC) and was honored to receive the highest funded project under the National Research Programme for Universities (NRPU). She acts as a Chairperson of the Department of Food Science at her institution.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"239057",title:"Dr.",name:"Rabia Shabir",middleName:null,surname:"Ahmad",slug:"rabia-shabir-ahmad",fullName:"Rabia Shabir Ahmad",profilePictureURL:"https://mts.intechopen.com/storage/users/239057/images/system/239057.jpg",biography:"Dr. Rabia Shabir Ahmad has a strong academic, teaching, and research background. She has a number of credits and honors in her career which comprises brilliant successes. In every competitive environment, she has proved herself the best. During her academic carrier, she secured a merit scholarship from the University of Agriculture, Faisalabad for her Bachelors and Masters degrees, and received a PhD Indigenous Scholarship during her PhD study. She played an important role in the establishment and development of the Institute of Home and Food Science at GC University, Faisalabad, and developed and taught courses to graduate and post-graduate level students both in food science and technology, and human nutrition and dietetics. During her stay at the Government College University Faisalabad, she won various HEC-funded research projects and was also honored to receive the highest funded project under the NRPU scheme from HEC. Along with her teaching and research supervising responsibilities, she has also been working as the reviewer of several journals, and has published numerous research papers in highly impacted international and national journals. 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From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"73333",title:"Lubricant and Lubricant Additives",doi:"10.5772/intechopen.93830",slug:"lubricant-and-lubricant-additives",body:'\nLubricants have been in use for hundreds of centuries and are essential to our survival. Natural lubricants such as saliva and synovial fluid lubricate the food for easy mastication and reduce wear and tear of our joints respectively. Cooking oils prevent sticking of food onto frying pans and baking trays at the same time as conducting heat. Ancient Egyptians used lubricants to slide large stone blocks for building the great pyramids while the Romans used lubricant on the axles of their chariots [1]. Ancient lubricants were plant and animal based natural oils. With the onset of industrial revolution and our reliance on metal-based machinery and engines, petroleum-based lubricants witnessed a growth.
\nModern lubricants are far more complex and perform various other functions in addition to lubricating such as cleaning, cooling, and sealing. The primary function of most lubricants is to reduce friction and this property is known as lubricity. A lubricant can be used in solid form, semi-solid, liquid form or gaseous form. Examples of solid lubricants are graphite and Molybdenum disulphide (MoS2), semi-solid lubricants are greases, and liquid are automobile engine oil. Depending on the requirements of a said application, the physical state of lubricant is chosen. For example, in space environments where liquid lubrication is not feasible due to vacuum, solid lubricants are chosen. Air bearing are preferred in applications in machine tool applications where precision is of primary importance such as cutting and finishing of optical lenses. Greases are used where a liquid oil would not remain in position due to its tendency to flow or when a sealing action is needed to prevent water-ingress in addition to lubrication. Today’s lubricants are designed and packaged to meet specific requirements for specific applications by lubricant formulators. The lubricant for automobile transmission and drive train has different requirements to satisfy compared to lubricant for an internal combustion engine or turbines. Further depending upon the type of turbines viz. gas, steam or hydraulic, the lubricant needs to be designed.
\nTypically, industrial lubricants contain 70-90% base oils and the rest is additives [2]. Base oils impart primary vital properties of the lubricant such as viscosity, viscosity stability, thermal stability, solvency, low temperature flow and volatility, oxidation stability. Additives have been used in lubricating oil since the 1920s and the demand for lubrication has resulted in continuous growth in the size of the market (USD 14.35 billion in 2015) with huge investments in research and development to design and formulate superior lubricants that meets present and future environmental regulations and consumer expectations. Despite this, lubricant formulation has mostly remained an art. This is because blending a new formulation for optimizing viscosity and obtaining optimum performance through performance tests for friction and emissions is much easier than testing for parameters, such as impact on engine wear, sludge build-up and piston cleanliness which require long duration engine tests. For example, during the development of Castrol’s engine oil, ‘Edge with Titanium Fluid Strength Technology’, over 2400 unique formulations were engine-tested for an equivalent of 1.9 million miles.
\nTo understand the need for additives, one must understand the implication of the Stribeck curve shown in Figure 1. Machine elements such as engine bearings work in hydrodynamic lubrication regime where the major function of the lubricant is to maintain its viscosity at all temperatures while ensuring a thick fluid film to keep the two contacting surfaces in relative motion separated at all loads and speeds. Rolling element bearings work on elasto-hydrodynamic lubrication (EHL) where the contacting surfaces deform elastically and there is a very thin film separation. Cams and tappets work in the boundary lubrication regime where there is substantial metal to metal contact. And in the reciprocating motion of engine piston rings, all four kinds of lubrication regime occur.
\nStribeck curve.
Classical lubrication theory assumes that a lubricating oil is a Newtonian fluid with a fixed viscosity and the contacting surfaces to be rigid. George Osborne Reynolds approached the fluid film hydrodynamic lubrication using mathematical and physical approach (1886) to predict friction, film thickness and load carrying capacity [3]. In the real world, oils undergo shear thinning and behave as a non-Newtonian fluid due to heat and pressure developed at the contact. Furthermore, real surfaces are rough and can undergo elastic as well as local plastic deformation under fluid pressure. The EHL theory was developed by H.M. Martin (in 1916) and later by Ertel (in 1939) and Grubin (in 1949), Petrusevich (in 1951), Dowson and Higginson (in 1959), Dowson and Hamrock (in 1977) for predicting traction, load carrying capacity and film thickness in heavily loaded contacts [4]. The non-Newtonian behavior of thin films under high pressure and lower rolling speeds has guided lubricant formulators to consider the shear-stress/shear-strain behavior, pressure-viscosity dependence of the lubricant as these are closely linked to the molecular properties of the oil composition. On the Stribeck curve, when the speed is very low, the friction is governed by the chemistry of the lubricant molecules i.e. the molecular structure and orientation. It was W. B. Hardy (in 1920), who coined the term boundary lubrication and later with Ida Doubleday (in 1922) established the basic concepts of boundary lubrication theory [5]. The boundary lubrication properties such as friction and film thickness at the interface of two surfaces are affected by the force fields of molecules in relation to their structure and polarity. Hence, formulators add boundary film additives to reduce friction in this regime [6]. Additives must also reduce wear, as in this regime, the two surfaces in relative motion are in contact hence prone to significant wear. Furthermore, additives need to counter the side effects of continuous as well as intermittent use of the lubricant such as they need to control heat, deposit formation, prevent foam formation, prevent fouling and corrosion due to water ingress, prevent wear, increase film strength under concentrated contacts, control greenhouse gas emissions. Therefore, as high as 30% content of modern automotive lubricants are chemical additives while some industrial oils may only contain 1% or less.
\nA typical petroleum-based oil with no additive is called a base oil or base stock [7]. The generation of base stock starts with the identification and selection of a good petroleum crude followed by atmospheric distillation, vacuum distillation and solvent processing. Solvent processing has two processes viz. solvent extraction, and solvent dewaxing where undesirable molecules are separated where as in hydroprocessing undesirable molecules are converted to desirable ones. Hydroprocessing a general term for conversion of less desirable crude fractions into good quality feedstock using catalyst and hydrogen at high temperature and pressure. This can be categorized as hydrofinishing, hydrotreating and hydrocracking according to increasing order of severity. Another process called hydrodewaxing is required to remove long chain linear paraffins, isomerize straight chains to branched chains which improves the pour point. Only 10 % of crudes are converted to base stocks for lubricants, hence the refinery owners call the shots regarding the choice of crudes to be converted to base oils while balancing the cost, yield and demand relative to all the other refinery products. However, to satisfy lubricant performance demands under severe operating conditions, high quality base stocks are needed. The lubricant manufactures (refinery owner can also be lube manufacturer) buy these base stocks and other chemical compounds and formulate their lubricant for example SAE15W40 or ILSAC GF-5, meeting standards set by Original Equipment Manufacturers (OEMs), professional bodies, and international institutions like American Petroleum Institute (API), International Lubricant Standardization and Approval Committee (ILSAC), Society of Automotive Engineers (SAE) to name a few.
\nOwing their origin to petroleum crudes, lubricant base stocks are also mixtures of long chain hydrocarbons containing three types of chemical groups. i.e. paraffins, naphthenes and aromatics. The paraffins can be further classified into branched or straight chains. The chain length and branching affects the melting point and crystallization temperature of the paraffins. During the production of lube base stock, most of the unsaturated bonds, paraffin wax and sulfur content are removed, however depending on the severity of hydroprocessing, some wax, unsaturates and sulfur may remain. Base Stocks have been classified into 5 categories by the API according to the presence of saturates, sulfur content and viscosity as shown in Table 1. Group I, II and III are derived from petroleum crude while Group IV is reserved for Polyalphaolefins (PAO) which are synthesized from gaseous hydrocarbons. Group V is for all other base stocks that are not included in other four groups such as mineral based napthenics, synthetic esters, polyglycols, silicones, polybutenes, phosphate esters etc. These oils are designed for severe performance requirements.
\n\n | Type | \nSaturates (%) | \nSulfur (%) | \nViscosity index | \n
---|---|---|---|---|
Group I | \nSolvent refined | \n<90 | \n>0.03 | \n80 to <120 | \n
Group II | \nHydrofinished | \n≥90 | \n≤0.03 | \n80 to <120 | \n
Group III | \nHydrocracked | \n≥90 | \n≤0.03 | \n≥120 | \n
Group IV | \nPolyalphaolefins | \n— | \n— | \n— | \n
Group V | \nSynthetics and naphthenics and other stocks not included in Group I, II, III or IV | \n— | \n— | \n— | \n
API base stock classification.
Group I base oils are processed by solvent processing in which wax and multiring aromatics are removed while some sulfur and aromatics remain. These base oils are typically used in marine and diesel engine oils, heat transfer oils, hydraulic oils, conventional greases, industrial gear oils, and machine tool oils. These oils have high solvency, very high viscosity but poor viscosity index. The maximum operating temperature is 93°C.
\nGroup II base stocks have undergone catalytic conversion to remove wax, aromatics and sulfur compounds. These base oils are used as automotive engine oil, automatic transmission fluid, gear oils and turbine oils. The maximum operating temperature of these base stocks is 121°C.
\nGroup III base oils are similar to Group II oils but have a higher viscosity index. The severity of hydroprocessing removes any ring structures, as a result, its solvency is poor. These base oils are used in premium passenger vehicles, automatic transmission fluids, and food grade lubricants. The maximum operating temperature is 121°C.
\nGroup IV oils are synthetically produced Polyalphaolefins from low molecular weight organic material. They have uniform molecular structures. They provide excellent high temperature performance and oxidation stability and therefore used in high performance engine and gear applications, heavy duty industrial compressor, power transmission fluid, hydraulic fluid, heat transfer fluid and in bearings as greases or liquid or lubricants. A wide range of viscosity grades of PAOs can be produced by varying the number of olefin molecules linked together. PAOs have a maximum operating temperature of 132°C.
\nGroup V base stocks can be either naphthenic or synthetic in origin. The naphthenics provide very good low temperature performance and hence are used in applications which operate in a narrow temperature range such as transformer oil, process oils, grease. These oils have high solubility and available in a wide range of viscosities. The synthetics on the other side vary widely in their types and properties. These include polyglycols, silicones, polybutenes, organic esters, phosphate esters and they are used as compressor oils, brake fluids, heat transfer oils, aviation engine oil. Their maximum operating temperature is dependent on the nature of their molecular structure. Silicon oils are known to have a maximum operating temperature of 232°C.
\nFinally, Group II+ and Group III+ are two types of base stocks that are not included in the original API classification. The plus refers to increase in viscosity index (VI) in the higher limit of the API specification. Group II+ has VI minimum between 110 and 115 and Group III+ has minimum VI somewhere between 130 and 140.
\nAs mentioned previously, mineral oils are mixtures of hydrocarbons containing paraffins, naphthenics and aromatics of various structures and carbon numbers ranging between 20 to 40+, at varying amounts depending on their degree of refining and processing. Typical composition of various API groups have been identified, but their exact structures are still not known. There is a considerable variability in the performance among the mineral origin base oils owing to the source of the crude. Therefore, lubricant manufactures conduct performance tests on their formulations to ascertain satisfactory performance with any new base stock in the same API category.
\nThe primary function of Lubricant additives is to improve the properties of the base stock under different operating conditions and the high performance requirements of any machinery. Lubricant additives are chemical components that need to blend well with the base oil to function as a single fluid. Additive manufactures often sell several additives combined into an additive package and diluted with a base oil at a higher concentration. The additive package is then dosed into the lubricant blend by the lubricant manufacturer at an appropriate treat rate to give the desired performance. The concentration of various additives is constrained by various factors such as their primary function (for example dispersancy, wear protection and so on), their synergetic or antagonistic behaviour with other additives, and regulations set by industry bodies.
\nIn a nutshell, lubricant additives can be categorized into various kinds based on their general roles of performance improvement and service life extension. First category are additives that impart new properties to the lubricant also known as surface protective additives. Examples include antiwear additives, extreme pressure additives, corrosion inhibitors, detergents and dispersants. The second kind of additives enhance the existing properties already present in the lubricant hence known as performance additives. Viscosity index improvers, viscosity modifiers, friction modifiers, pour point depressants belong to this type. The third type of additives known as lubricant protective additives, are the ones that counteract the negative effects or changes that take place during the service life of the lubricant. These include antifoamants and, antioxidants. In this chapter only the major types of lubricant additives are discussed. Other additives such as demulsifiers, emulsifier, biocides may be added depending on the intended applications.
\nAs the name indicates, these are additives that reduce the pour point of the lubricant, i.e. the lubricant remains in liquid state and maintains its fluidity (pourability) at lower temperatures than without these additives. Usually as temperature decreases, paraffin molecules in the oil start to crystallize as wax (below 50°C) and the oil loses its ability to flow by gravity or to be pumped under pressure. This also affects the viscosity of the oil. Additives such as alkylaromatic polymers and polymethacrylates prevent wax crystal growth by modifying the interface between the wax and the oil molecules, to a certain extent thus lowering the pour point by about 20–30°F (11–17°C). These are present up to a fraction of a percent in all paraffin-based lubricants that lubricate machine elements such as bearings, gears exposed to cold start and cold (winter) operating temperatures. Modern multi-grade engine oils/motor oils composed of partly synthetic oil and partly mineral oil along with these additives, have pourpoints as low as −32°C.
\nViscosity index improvers (VII) also known as viscosity modifiers are additives that prevent the oil from losing its viscosity at high temperatures which is a natural tendency of any liquid. These additives are available in all shapes and sizes and quality [8]. Polymethylmethacrylates, olefin copolymers, hydrogenated poly(styrene-co-butadiene or isoprene), esterified polystyrene-co-maleic anhydride are commonly used VIIs. The large oil soluble flexible polymer molecules uncoil and spread out as temperature increases thereby increasing the viscosity as shown in Figure 2. Furthermore, their numerous branches entangle with those of other neighboring molecules. By doing this, these macromolecular structures can trap and control smaller oil molecules, thus increasing the viscosity of the lubricant.
\nMechanism of VII.
Permanent and temporary shear thinning of VII-thickened formulations can also occur depending upon the quality of the VII. In heavy duty application, due to the large compressive pressure between the two mating surfaces, VII polymer molecules, tend to align with each other and get “squashed” or even get chopped to small pieces under high shear conditions. When the polymer coils elongate and become aligned in the direction of the flow the viscosity temporarily drops resulting in reduced oil film thickness. After the lubricant leaves the contact between the mating parts, the polymer coils return to their original shape and the viscosity of the lubricant returns to normal. This phenomenon is referred to as temporary shear-thinning. However, under further high shear rates, the long and flexible polymer chains can be cut or ruptured or pulled and ripped apart into smaller chains by molecular scission. Unfortunately, once this has occurred, the broken polymer chains cannot re-form into the single large chain and this causes the oil to permanently lose viscosity leading to a reduction in oil film thickness, oil film failure and an increase in wear. This phenomenon is referred to as permanent shear-thinning.
\nAnti-wear additives are additives that prevent two-body wear of the metallic countersurfaces in the boundary lubrication regime where the film thickness is small and there is asperity - asperity contact. These additives are polar in nature which enables them to attach to the metallic surfaces followed by tribochemical or mechanochemical reactions to form an anti-wear film. This newly formed film undergoes wear and formation at the top layers thus protecting the underlying metallic surface. As these additives form films by chemical reactions, they get used up and the amount of antiwear additives present in the lubricant reduces with time. These are typically phosphorous compounds. Zinc dialkyldithiophosphate (ZDDP) is the most common, the most researched and has been used since the 1940s [9]. Its use has been reduced in passenger vehicles in the last decade due to zinc metal causing poisoning of the catalyst in the exhaust gas catalytic convertor. ZDDP also provide antioxidant and corrosion-inhibition properties to the lubricant. Owing to the multi functionality of ZDDP, finding its replacement has been challenging because molybdenum-based additive such MoDTC (molybdenum dithiocarbamate) or MoDDP (molybdenum dithiophosphate) molecules cannot work as antioxidant. On the other hand, ash-less antiwear additives such as hindered phenols and amines are very expensive and are required in larger quantities. Till date, ZDDP is considered as the most cost-effective antioxidant and antiwear additive available, and the alternatives are currently very expensive.
\nAntioxidants or oxidation inhibitors prevent the oxidation of the components of the base oil there by increasing the life of the lubricant. Oxidation of the lubricant molecules occur at all temperatures but at higher temperatures, it is accelerated. The presence of wear particles, water, and other contaminants also promote Oxidation of the lubricant molecules which then leads to formation of acids and sludge. The acids may further cause corrosion in the metallic parts while the sludge formation increases the viscosity of the lubricant. Almost every lubricating oil and grease contains antioxidants and examples include Zincdialkyldithiophosphates, hindered phenols, sulphurized phenols, and aromatic amines. These compounds decompose peroxides and terminate free-radical reactions that occur in the lubricant. These are sacrificial in nature hence their quantity gets reduced with time.
\nDefoamants or antifoaming agents are additives that prevent the lubricant from forming a foam and speed up the collapse of the foam if it does form. Foaming occurs because of constant mixing of the oil with air or other gases leading to air entrapment. Foam disrupts cooling of parts as it is not a good conductor of heat. It reduces the load carrying capacity and the lubricant flow leading to excessive engine wear. Silicone polymers such as polymethylsiloxane at a few parts per million and organic copolymers such as alkoxy aliphatic acids, polyalkoxyamines, polyethylene glycols, and branched polyvinyl ethers, at higher concentration are widely used in mineral oils. The antifoaming agents are essentially insoluble in the lubricant hence they need to be finely dispersed in the lubricant. These droplets attach themselves to the entrapped air bubbles and aid in forming bigger bubbles (via coalesce). The larger bubbles rise readily to the surface followed by bursting to release the trapped air. Bursting occurs by thinning of the air bubble film as the additive spreads due to its low surface tension.
\nFriction modifiers are used in engine oil and transmission oil to alter the coefficient of friction that would be experienced between the sliding parts when only the base oil is present. Friction reduction results in improved fuel economy. Organic and sulfurised fatty acids, amines, amides, imides, high molecular weight organic phosphorus and phosphoric acid esters are added to the range between 0.1 and 1.5% in finished lubricants as friction modifiers. Glyceryl monooleates and Molybdenum compounds such as MoDTC and MoDTP also function as friction modifiers. They preferentially adsorb very strongly on to the metallic surface. The head of the friction modifier is attracted to the metal surface and the long tail with at least 10 carbon atoms remains solubilized in the oil as shown in Figure 3 [10]. The chemical structure and the polarity of the molecules play a major role in the friction reduction. Ionic lubricants [11], a class of ionic liquids that are room-temperature molten salts consisting of cations and anions are also very good surface additives. The polarity of head group provides for strong surface adsorption. The physical, chemical and tribological properties of ionic liquids can be tailored to suit a wide variety of applications ranging from its use as polymer brushes in biological application, or as water soluble or oil soluble lubricant additive.
\nAdsorption of polar headgroups onto metallic surface.
Detergents keep surfaces free of deposits and neutralize corrosive acids formed due to oxidation. These molecules are chemical bases consisting of a polar substrate and a metal oxide or hydroxide [12]. Metallo-organic compounds of calcium and magnesium phenolates, phosphates, salicylate and sulfonates are recommended. Overbased detergents are used in marine engine lubricants to neutralize large amounts of acidic components produced by fuel combustion or oil oxidation. Ash (burning of organometallic species) and soot particles (largely carbon with sulfur adsorbed) is formed by burning of the oil in internal combustion engines. Ash can then form unwanted residues at high temperatures or simply deposit on surfaces. The deposit precursors particles are insoluble in the oil and have greater affinity for detergent molecules. The additive molecules cling to the surface of the particle and envelop it thereby also acting as dispersants and prevent those particles to agglomerate and to later settle as deposits. A detergent additive is normally used in conjunction with a dispersant additive.
\nDispersants are used mainly in engine oil along with detergents to keep engines surfaces clean and free of deposits [12]. Dispersants keep the insoluble soot particles and the precursors of deposits in the internal combustion engine finely dispersed or suspended in the lubricant even at high temperatures. These suspended particles are subsequently removed by oil filtration or oil change. Thus, dispersants minimize damage to engine surfaces and formation of high temperature deposits. Generally, polymeric and ashless dispersants are used today such as polymeric alkylthiophosphonates, alkylsuccinimides, succinic acid esters/amides, and their borated derivatives as well as organic complexes containing nitrogen compounds.
\nCorrosion and rust inhibitors are additives that reduce or eliminate rust (corrosion of iron and steel) and corrosion by neutralizing acids and forming a protective film, either adsorbed or chemically bonded on the metal surfaces. Preferential adsorption of polar constituent on metal surface forms the protective film that prevents corrosive materials such as organic acids from reaching and attacking the metal. These are usually compounds having a high polar attraction towards metal surfaces such as succinates, alkyl earth sulfonates, metal phenolates, fatty acids, amines as well as zincdithiophosphates. Some of these inhibitors are specific to protecting certain metals. Hence, an oil may contain several types of corrosion inhibitors.
\nExtreme Pressure additives are required to reduce friction, control wear and prevent severe surface damage in heavy duty application of gears and bearings at high temperatures and pressures. They are also known as antiscuffing additives. They react chemically with metallic surfaces to form a sacrificial surface film that prevents the welding and subsequent seizure of asperities at the metal-to-metal contact. Additionally, they contribute to smoothing of the surfaces as these are formed at contact asperities and the load is then distributed uniformly over a greater contact area, thus reducing the severity of wear and ensuring effective lubrication. Effectiveness of EP additives relies on their reactivity and their ability to readily form thick surface films at high loads and high contact temperatures that are created at the mechanical contacts. These additives usually contain sulfur and phosphorus compounds and chlorine or boron compounds. Ashless EP additives such as dithiocarbamates, dithiophospates, thiolesters, phosphorothioates, thiadiazoles, aminephosphates, phosphites may be preferred in some applications where chlorine may cause corrosion.
\nOther additives such as demulsifiers, emulsifier, biocides are added to meet specific requirements. Emulsifiers are used as a binder between oil and water molecules in oil-water-based metal-working fluids to help create a stable oil-water emulsion. Without the emulsifier, oil and water will separate out from each other due to differences in specific gravity and interfacial tension. On the other hand, demulsifiers are used to separate oil-water emulsions. Demulsification and removal of the aqueous phase from the oil-based lubricant minimizes harmful effects such as corrosion, foaming and cavitation from occurring. Biocides may be added to water-based lubricants to control the bacterial growth.
\nLubricating grease are a class of lubricant that do not flow like a fluid but bleed (release oil) when squeezed between contacting surfaces. They have a gel like consistency and can be described as a solid or semifluid like material and in some cases can be used in vertical or overhead applications because they can have good drip resistance due to their Non-Newtonian rheological properties. They are particularly useful in applications that are sealed for life; for example bearings and remote gearboxes. Functionality of greases include sealing out contamination and water ingress, prevent corrosion, compatible with polymers and elastomers, provide antiwear and extreme pressure load protection while reducing friction. Greases have three main components: fluid, thickener and additives [13].
\nA typical grease consists of 75-95% fluid base stock, 2-25% thickener and 0-25% additives. The base stock is chosen based on the required applications. Hence, the fluid can be petroleum based for most automotive and industrial application, synthetic based for low and high temperature application or may be wax based (no flow) for high load caring capacity. The thickeners are metal soaps which are created using the fundamental reaction of an acid and a base. Calcium based soaps have been the simplest and earliest used thickeners with a maximum operating temperature between 60 and 70°C. In the last decade, calcium sulfonate greases, polyurea greases, aluminum complex greases, lithium complex greases, sodium complex greases, and clay-based greases have received general acceptance due to their higher service temperatures of more than 150°C. However, each having their own set of pros and cons. For example, calcium-based greases do not perform well over a wide range of temperature, sodium based have deteriorated performance in the presence of water. Clay and polyurea based greases are used for high temperature (service temperatures of 190-220°C) and application that have limited relubrication access. The third component of greases are additives. Commonly used additives are listed below.
Antiwear additives
Antioxidants
Extreme Pressure additives
Friction Modifier
Rust and corrosion inhibitor
Tackifiers (adhesive agents)
Odorants (perfumes)
Dyes
Tackifiers are additives that increase the adhesive property of the grease or the lubricant. They prevent the lubricant from flinging off the metal surface during rotational movement. To be acceptable to the manufacturers and the end users the greases must be free of offensive odor and have a desirable color. Therefore, odorants and dyes are added to the grease. Although these have little effect on the grease performance, their appeal to senses has an impact on the product selection.
\nGreases have the unique ability to incorporate liquid as well solid additives. Solid additives such as molybdenum disulphide (MoS2), graphite, hexaboron nitride and polytetrafluoroethylene (PTFE) in the form of fine dispersed powder (nano and micro particles) have been used in lubricants and greases to provide ultralow friction and wear protection. Solid additives provide a physical separation between two contacting surfaces when fluid is unable to provide load support. The lattice structure of these solid lubricants plays an important role in transferring a thin low shear layer on the metal surfaces especially where load is high, and speed is low.
\nSolid lubricants on their own are vital to niche applications such as space missions, satellites release and deployment mechanisms. Vacuum and microgravity of space eliminates the use of liquid lubricants. The importance of a thin film of solid lubricant can be emphasized on the fact that the success of an entire mission can be compromised if the, receiver and transmitter antennas or solar arrays packed securely during launch fail to release smoothly with precision due to extreme friction of a deployment mechanism. Solid lubricants can be used at low temperatures as well as at high temperatures where the liquid lubricant may solidify or vaporize respectively. Even at extreme pressures where liquid lubricant will be squeezed out, solid lubricants are used. However, solid lubricants are not limited to extreme conditions [14]. A wide variety of low friction coatings are used in various engineering applications that require high electrical and thermal conductivities, low wear rates and high lubricity at all operating temperatures. Newer engineered coatings have increased complexity and have transitioned from single or multi component structures to nanostructured and functional gradient structures.
\nThe most recognized polytetrafluoroethylene (PTFE) coating is Teflon® discovered in 1938 at DuPont. These are highly linear fluorocarbon molecules. They offer a low friction surface with moderate wear. They have low chemical reactivity and low surface energy. PTFE on its own performs best at low loads and wears rapidly at higher loads; hence they need reinforcements to increase strength and load bearing capacity. Such materials are called composite materials.
\nCarbon based materials such as graphite in both micro and nano forms is a popular solid lubricant additive whereas diamondlike carbon (DLC) makes excellent low friction coatings. However, they have different mechanisms of friction reduction. Graphite has hexagonal crystal structure which has the intrinsic property of easy shear. DLC exhibits high hardness and low friction due to an amorphous structure that combines graphitic and diamond phases. They can be doped with hydrogen or nitrogen for achieving desirable properties. Recently, a series of patents on superlubricity of nano-diamonds and graphene films have been filled [15].
\nMolybdenum disulphide are transition-metal dichalcogenides like Tungsten disulphide (WS2) work on the mechanism of interlamellar shear between covalently bonded hexagonal basal planes like that of graphite. Their performance is affected by moisture content [16].
\nApart from the advantages mentioned earlier, solid lubricants also have a few disadvantages. They have less ability to carry away heat and contaminants away from the contact. They have poor self-healing properties, and they are not easily entrained into tribological contacts.
\nAfter the selection of a suitable lubricant, its application method i.e. its delivery to the mechanical components such as gears, bearings, cams, tappets, chains, guideways and couplings of a machine or engine in correct quantity is considered. Liquid lubricants are applied to the machine elements by two methods. First type is called ‘all loss method’ while second type is called ‘reuse method’ [2]. In all loss method a small quantity of lubricant is applied periodically and the lubricant after use, gradually leaks away to waste. In reuse method, elaborate lubricating systems are designed to feed the required quantity of lubricant to various machine elements of the system. The lubricant after leaving the machine components is collected, cooled then filtered and recirculated to lubricate the machine components again. Most open gears, ropes, guideways, chains and rolling element bearings (except sealed for life rolling element bearings) are lubricated by all loss method. Nearly all grease lubricated elements also are lubricated by this method. Some devices used for all loss lubrication method include hand-held oiling device, grease gun, drop feed cups, wick feed cups, wick oilers, pad oilers, mechanical force feed lubricators, airline oilers and automatic or semiautomatic spray units. Reuse method of lubricant application has the oil circulating through a network of pipes that is pressurized by a pump or aided by gravity. However, closed oil sump systems employ splash oiling, bath oiling or ring oiling methods. Centralized lubricant application systems can reduce the quantity of lubricant usage as well as labour costs. Automobiles use an oil mist lubrication system to lubricate various machine elements of the engine and drive train. Considerations with regards to the lubricant characteristics and composition are required while designing a compatible system. Oil condition monitoring and routine checks are vital for the longevity of the machine elements lubricated by any method.
\nSolid lubricants on the other hand are applied as powder dispersed in a liquid lubricant or in a solid phase matrix. They can be also applied as a thin film coating. The coatings are made by various methods such as dip coating, thermal spraying, and cold. More robust ways of coating includes chemical vapor deposition or physical vapor deposition methods. Electrochemical processes are used for producing coatings of polymer-based solid lubricants and their composites.
\nLubricant analysis primarily refers to the characterization and evaluation of the lubricant for various physical, chemical and performance properties in all stages of its life cycle. For solid lubricants and coatings, the analysis includes determining the composition and structure by using a range of spectroscopic and microscopic observation methods as well as measuring the coating consistency and thickness applying non-destructive evaluation techniques. Standardized tribological lab tests are used to evaluate their performance. Once a candidate material is identified, larger-scale bench testing, such as engine tests, are conducted.
\nFor liquid lubricants, characterization is done to a much larger extent due to the large variety of applications and the implication of chemistry of the formulated oils on the machine elements performance and the overall performance of the entire equipment/machine or engine. The lubricant analysis can be classified as physical and chemical characteristics evaluation, Performance test evaluation and Engine Test evaluation as summarized in Table 2 [2]. Additionally, end users also develop their in-house in-service lubricant analysis to monitor and maintain the condition of the lubricant. The objective of such analysis is to ensure optimum performance, achieve expected life of the equipment as well as the lubricant flowing through it.
\nPhysical and chemical characteristics | \nPerformance test evaluation | \nEngine test evaluation | \n
---|---|---|
Color | \nOxidation Tests | \nOxidation stability and bearing corrosion protection | \n
Density and API gravity | \nThermal Stability | \nSingle cylinder high Temperature tests | \n
Carbon Residue | \nFoaming Tests | \nMulti cylinder high temperature tests | \n
Flash point | \nCorrosion and Rust Protection Test | \nMulti cylinder low temperature tests | \n
Neutralization Number | \nEP and Antiwear Test | \nRust and corrosion protection tests | \n
Total Acid Number | \nEmulsion and Demulsibility Test | \nOil Consumption rates and volatility | \n
Total Base Number | \n\n | Emission and protection of emission control systems | \n
Pour Point | \n\n | Fuel Economy | \n
Sulphated Ash | \n\n | \n |
Viscosity | \n\n | \n |
Volatility | \n\n | \n |
Evaluation techniques and testing.
Current automotive lubricants are optimized for internal combustion engines and drive trains. Electric vehicles (EVs) which use electric motors possess new challenges of lubrication such as high-power density of the small gear box which require efficient cooling. Hydro lubricants and synthetic gear oils are excellent candidates for such application but may pose sealing issues which requires innovative solutions. Lubricants are also required in the rolling element bearings of EVs that must stop electro-erosion caused by high frequency high energy discharges by being conductive. Ionic liquids are having shown good performance in preventing built up of potential [17].
\nThe future for lubricants formulation, manufacturing and end-use is oriented towards efficiency in terms of cost and time, customized and optimized for each individual tribo-system and run reliably for even longer drain interval time. New industrial lubricants must meet stringent regulations and guarantee ecological sustainability, and climate change actions. Hence, new lubricants will contribute to ‘Green Tribology’.
\nTribology of lubricants plays a significant role in technology and economics of industrial development. As the fourth industrial revolution ‘Industry 4.0’ (combines automation with the internet of things) is in progress, several concepts can be extended to lubricant and lubricant additive development and evaluation. For example new electronic smart sensors can be used for lubricant analysis and condition monitoring. The information of various performance parameters can be stored as data and transferred to the stakeholders and decision-making points using the information and communication technologies involved in Industry 4.0. such as internet and wireless connections to and via several electronic devices. Continuous monitoring can also aid in corrective measures. In-service lubricant performance testing and evaluation generates big quantities of data from various equipment and sensors. Therefore, ‘Big Data’ concepts can be applied in several ways. One such example can be combining lubricant data with machine data, another can be correlation study of amount of soot produced, change in oil viscosity, friction, wear of an engine. The benefits of Industry 4.0 include shorter lubricant development time, reduced number of trials, lubricant performance prediction through chemical and physical modeling and simulations. This will transform product development process from being empirically driven to using data and simulation driven approach.
\nLubricants are vital for the tribological life of the machine elements. As modern machine elements are required to perform in heavy-duty applications in a wide range of environments, newer, better and environmentally sustainable lubricants are required to be designed. Lubricant additives are therefore considered as lubrication engineering design components. Lubricant additives are designed and optimized to meet the performance requirements of the equipment or engine. Various types of lubricant additives and their functional properties were discussed. They are designed to provide oxidation resistance, high temperature viscosity, energy and fuel efficiency among others. Lubricant or grease therefore are a complex mixture of several components blended carefully together to meet the performance requirements. The different components can have synergistic or antagonistic effects due to chemical interactions or competition at the metal surface or among themselves. Therefore, formulation of lubricants requires considerable expertise and expensive performance testing. Green Tribology and Industry 4.0 era will steer the lubricant development, use and disposal.
\nThis chapter could not be published without the exceptional support of Prof. Paul B. Davies (University of Cambridge, UK) and substantial industry inputs and scrutiny by Mr. Tony D. Smith (Castrol, UK).
\nIn the last years, novel and effective immunotherapies for patients with different tumor types are becoming clinically important, because of the remarkable clinical efficacy observed with several immune checkpoint inhibitors such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and the programed death receptor 1 (PD-1) or its ligand (PD-L1) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. Whereas anti-CTLA-4 antibodies (ipilimumab and tremelimumab), anti-PD-1 antibodies (nivolumab and pembrolizumab), and anti-PD-L1 antibodies (atezolizumab, avelumab, and durvalumab) have produced remarkable results, increasing the survival prognosis in many cancer types, it is still unknown why some tumors do not respond to or relapse after this type of treatment. In this way, increased observations suggest that tumors rich in tumor-associated immune cells (TAICs) may respond to therapies targeting immune system inhibitory or stimulatory mechanisms, and tumors with non-TAICs may require additional interventions aimed at promoting optimal inflammation and innate immune activation in the tumor microenvironment [13, 14, 15]. Indeed, characterization of different immune checkpoints as well as tumor microenvironment in patients with cancer has become a fundamental step in providing evidence for the presence of distinct immunologic phenotypes, based on the presence or absence of various immune cells [1, 16, 17] that can predict the response to the therapy. In this way, the study of immune checkpoints and TAICs and their interaction prompt the need for multiplexed analyses of tumor tissues. To address this need, in the last years, multiplex imaging platforms have emerged as an important tool to provide critical information about cancer microenvironment, prognosis, therapy, and relapse [18, 19, 20, 21, 22]. Different components in the tumor microenvironment can be examined simultaneously using multiplex methodologies, providing insight into biological cross-talk present at the tumor-host interface and from subcellular levels to entered cell populations. In addition, the precision of these new techniques can be used to evaluate the special distribution of multiple biomarkers detected simultaneously, and their coexpressions or interactions between cells are becoming a essential tool to study tumor tissues [22] and to ultimately enhance disease diagnosis and better inform timely patient care [23].
Multiplex technologies are being used to identify the presence of multiple biological markers as immune checkpoint and TAICs on a single tissue sample [24]. The multiplex imagining techniques provide unique biological information that in many cases cannot be obtained by other imaging methods or by single immunohistochemistry (IHC) techniques. As mentioned, individual cells can be accessed with extraordinary fidelity equal to that achievable in the bulk population, such that even rare cell populations can be studied to understand their important role in translational research, and this knowledge can be applied in cancer prevention and treatment. In this chapter, we will discuss one of the most reliable and a very well-known methodology to identify simultaneous biomarkers in formalin-fixed, paraffin-embedded (FFPE) specimens as well as its imaging analysis platform as an important tool for potential application in future cancer immunotherapy biomarker discoveries.
Tyramide signal amplification (TSA) was described in the 1990s by Bobrow and colleagues [25, 26]. It is an enzyme-linked signal amplification method that is used to detect and localize low copy number of proteins present in tissues by conventional IHC protocol, using, most commonly, alkaline phosphatase or horseradish peroxidase (HRP) enzymatic reaction to catalyze the deposition of tyramide-labeled molecules at the site of probe or epitope detection. Tyramides are conjugated to biotin or fluorescent labels and revealed by streptavidin-HRP system [27, 28]. The HRP catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to electron-rich tyrosine moieties close to the epitope of interest on FFPE tissue. Tissue surfaces with anchored biotinylated tyramide must be further treated with fluorescent- or enzyme-tagged proteins that have a high affinity for biotin, such as streptavidin, before microscopic visualization [27, 28]. The detection of the proteins is increased more than ten times compared to standard biotin-based staining methods [29].
Perkin Elmer developed the Opal™ workflow (Figure 1), which allows simultaneous staining of multiple biomarkers within a single paraffin tissue section. Multiplex immunofluorescence (mIF) allows researchers to use antibodies raised in the same species, and different panels combined with different targets can be created using this technology [21, 27]. The approach, in the manual protocol, involves detection with fluorescent TSA reagents, followed by microwave treatment that removes the primary and secondary antibodies between cycles and any nonspecific staining that reduces tissue autofluorescence for each antibody cycle. The correct ordering of the different antibodies in a panel is still challenging and is only solved by trial and error to obtain a perfect staining. In the automated protocol using Leica Bond RX or another autostainer, the time of staining is reduced drastically when compared with manual staining. The possibilities for mIF are expanding our knowledge of tumor immune contexture (Figure 2) in different types of cancers. Mapping the tumor microenvironment and the predictive and/or prognostic value of immune checkpoint expression on malignant cells and TAICs has been carried out in patients with melanoma, lung cancer, breast cancer, gastric cancer, Hodgkin lymphoma, and others by mIF [30, 31, 32, 33, 34]. Similar to other multiplex techniques, in the TSA mIF method, our experience showed that the approach to different targets requires diligent optimization, first in conventional chromogenic IHC validation and then in the simplex IF, before mIF staining in control tissues. The use of specific, very well-standardized, and validated antibodies, as well as the careful use of other components, as right antibody titration, incubation time, and antigen retrieval during staining, is important to obtain good and reproducible results using different panels [27]. The use of very well-known control tissues during each staining and for each created panel that allows all the markers is important and essential to detect possible staining errors during the process in each mIF panel. Properties of the FFPE material, such as sample age, method of preservation, storage conditions, and tissue type, are very important factors to be considered to obtain high-quality mIF staining and good data. Pathologists play a key role in making sure that tissue samples collected are appropriate for diagnostic and research purposes. The tissues need to be processed adequately, that is, fixed in 10% formalin and stored in good conditions to avoid antigenic deterioration that can influence the process when targeting several proteins using this methodology. Type of tissue, is another important factor to be considered, sometimes as a limitation factor for a quality staining with this technique. We observed that some tissues that have abundant fat as breast tissue or cartilage in some type of cancers or bone component that were submitted a decalcification procedures, are more challenges during the mIF staining, showing frequently artifacts of staining like background, folds, caused by tissue detached and unspecific or not clear staining on the cells, causes by the decalcification procedures. Antibodies with very good performance in decalcified tissues are limited, and those need an exhaustive validation in IHC before creating a new panel to stain these samples. No less important, the size of the sample is another factor to be considered during mIF staining; small biopsies as core needle biopsies (CNBs) less than 1.0 × 0.2 cm are more challenging and have high probability to be lost during standard mIF staining than bigger tissues as whole sections (~1.0 × 1.0 cm). The minimum number of malignant tumor cells required for mIF marker analysis has not been well established and is another factor to be considered during staining and analysis. According our experience, samples with at least more than 100 malignant cells are preferred, to avoid errors in the interpretations of different markers, especially when the targets of study are malignant cells. Necrotic areas in more than 50% of the entire sample can compromise the quality of the staining and when compromise the quality of the sample and the staining need to be considered judiciously by the pathology as excluded criteria to preserve the quality of the analysis and data (Table 1). The quality control of pathology, as a first step, is essential to avoid wasted effort, resources, and funds of the laboratory and to preserve the high-quality data obtained by this methodology.
Tyramide signal amplification workflow. After primary antibody (Ab), the HRP-conjugated secondary antibody binds to an unconjugated primary antibody specific to the target/antigen of interest. Detection is ultimately achieved with a fluorophore-conjugated tyramide molecule that serves as the substrate for HRP. Activated tyramide forms covalent bonds with tyrosine residues on or neighboring the protein of interest and is permanently deposited upon the site of the antigen. The method allows for serial cycles of the primary/secondary antibody pairs, while preserving the antigen-associated fluorescence signal, making this process amenable to multiple rounds of staining in a sequential fashion.
(A) Vectra® and (B) Polaris™ scanner systems, (C) low magnification image, showing the selection of five intratumoral areas of interest to be analyzed, (D) composite image of lung cancer tissue showing seven color markers to identify different cell populations, (E) tissue segmentation (epithelial and stromal compartments), and (F) cell populations’ immune phenotyping.
Specimen type | Size (cm) | Viable tumor cells (N) | Necrosis (%) | Fat/cartilage/bone (%) | Adequacy for mIF staining* |
---|---|---|---|---|---|
Whole section | >1.0 × 1.0 | >100 | 0 or <10 | 0 or <10 of any component | 100% in our series |
Whole section | >1.0 × 1.0 | >100 | 0 or >10 | >50 of any component | 80% in our series |
Small biopsies | >1.0 × 0.2 | >100 | 0 or <10 | 0 or <5 of any component | 100% in our series |
Small biopsies | <1.0 × 0.2 | >100 | <10 | <50 of any component | 70% in our series |
Small biopsies | <1.0× 0.2 | <100 | >10 | >50 of any component | 50% in our series |
TMA (by core) | >0.1 | >100 | 0 or <5 | 0 or <5 of any component | 100% in our series |
Quality criteria’s samples for multiplex immunofluorescence.
A preliminary quality control to establish the samples by a pathologist is strongly recommended to optimize the preparation of tissue for multiplex immunofluorescence staining and ultimately to guaranty a quality data. Each case needs to be considered separately and can be influenced by several characteristics. In general, the quality of the samples including the fixation process of the FFPE tissues, storage and cutting procedures, will influence the quality of multiplex staining (*). There are, however, according to our experience, different tissue characteristics that need to be considered as challenges for staining and analysis, and these are considered sometimes as limitations of the staining. By understanding much better these tissue limitations, we can avoid wasted effort, resources, and funds of the laboratory as well as preserve the high-quality data obtained by this technique.
Although the methodology of TSA is available for FFPE material and can enable multiparametric readouts from a single tissue section, they sometimes have limited scalability and throughput, related to limited number of markers allowed per panel compared with other multiplex methodologies like imaging mass cytometry and multiplexed ion beam imaging [35, 36]. The scanner system (Figure 2) Vectra® [27] from PerkinElmer provides high quality of scanning with high-resolution and multiband filter cubes that provide greater flexibility associated with the multispectral camera, to match with the sample. The new generation of scanner Polaris™ (PerkinElmer) scan system supports multiple filters using tunable LED excitation, similar to confocal microscope, and the captured signals are assembled in a composite image [37]. After acquiring the panoramic low-magnification images at ×4 or ×10, the specimens can be sampled using different ROI sizes by the phenochart (PerkinElmer) software viewer to scan high-resolution images at ×20 or ×40. Although, the scanner system Vectra®-Polaris™ can capture different regions of interest (ROIs) using the filters and the multispectral camera at high quality resolution [36], it is still impossible to accelerate the process of scanning or scan the whole tissue section as a unique image for the analysis. The time for scanning the sample is variable and depends on the number of markers used in the panel, number of ROIs captured per sample, and size of the ROI and can take from minutes to several hours according these parameters [38] (Table 2). According our experience, the TSA staining system for mIF when combined with multispectral image analysis software, such as InForm (PerkinElmer), can provide a powerful tool for analysis of multiple markers in one single slide [21, 39]. However, there are many available software in the market that can be used for the analysis of mIF images generated by the InForm software from the Vectra®-Polaris™ scanner systems, and it is important to know that the InForm software is essential to generate the individual unmixed tyramide fluorochrome with a positive signal without noise or aberrant background staining and with high resolution performance across the different ROIs from the scanning systems [40]. For the analysis, image analysis software need to be accessible (Table 3), with easy automated capabilities of detection, including tissue segmentation, compartmentalization of the staining (e.g., nuclear, membranous, or cytoplasmic) (Figure 2), and spatial colocalization of cell distribution, critically important to study different markers included in different panels (Figure 3). In the same way, comprehensive evaluation using this different image analysis software is needed not only for clear antigen demarcation and good staining procedures but also for good interpretation of the results. Pathologists are very important and need to standardize the possible interobserved variation [41, 42] when using different image analysis platforms during the colocalization of proteins.
Magnification | Scanner | ROI (Vectra®)/(PolarisTM) (seven markers) | |
---|---|---|---|
Vectra® (Time/minutes) | PolarisTM (Time/minutes) | ||
4×* | ~9 | – | Panoramic view |
10×* | ~18 | ~13 | Panoramic view |
20× | ~6 | ~5 | 1×1 (669×500 μm)/(931×698 μm) |
20× | ~12 | ~14 | 2×2 (1338×1000 μm)/(1862×1396 μm) |
20× | ~22 | ~35 | 3×3 (2007×1500 μm)/(2793×2094 μm) |
40× | ~4 | ~8 | 1×1 (334×250 μm)/(465×249 μm) |
40× | ~10 | ~20 | 2×2 (669×500 μm)/(931×698 μm) |
40× | ~19 | ~36 | 3×3 (1004×750 μm)/(1396×1047 μm) |
Approximate time for image scanning using Vectra® or the PolarisTM scanner system.
Available only in Vectra®.
The time for scanning the sample is variable and depends on the number of markers used in the panel, number of regions of interest (ROI) captured per sample, and size of the ROI using Vectra® or PolarisTM system, as well as whether the system stores the image locally or in a server.
Vendor | Program name | Method | Availability |
---|---|---|---|
PerkinElmer | InForm | Color-based colocalization, tissue, cell segmentation | Licensed |
Definiens | Tissue Studio | Imaging segmentation, marker intensity measurement, and statistical analysis | Licensed |
Indica Labs | HALO | Membrane, colocalization, immune cell proximity, spatial analysis | Licensed |
Visiopharm | Visimoph Tissuemorph | Signal intensity, area, counting objects, statistical analysis | Licensed |
Spot Imagine | Spot advanced | Color-based colocalization | Licensed |
FARSIGHT | Nucleus Editor | Multichannel-based object identification/toolkit | Free |
NIH | Image J | Color-based, user interactive segmentation | Free |
HistoRx | AQUAnalysis | Signal intensity per unit area and per layer | Licensed |
CompuCyte | iCyte | Nucleus segmentation or phantom contouring, measuring associated signals | Licensed |
Image analysis software systems available for multiplex immunofluorescence.
Microphotographs of representative examples of multiplex immunofluorescence in tumor tissues using different markers, (A) lung adenocarcinoma, (B) lung squamous cell carcinoma, (C) malignant melanoma, and (D) lung squamous cell carcinoma. ×20 magnification.
Despite the evolution in the last years, in different levels of cancer research, concerning prevention, diagnosis, therapeutic options, and follow-up methods, cancer diseases are still the major public health problem worldwide [43]. Profiling immune cells is currently a powerful metric for tumor subclassification and predicting clinical outcomes. A great variety of cancer research screening tools is applied to diagnose tumors and has been established for different tumors. Simultaneous quantification of more than one biomarker at the same time has become more and more interesting in cancer research using different multiplex technologies. Multiplex TSA can allow different biomarkers in one single slide, targeting different systemic processes, such as inflammation, immunocheckpoints, angiogenesis, or cell death using tumor markers (Figure 3), to improve cancer prevention, diagnostic accuracy, and treatment. We demonstrated that this method can offer important advantages, such as high-throughput performance, low material requirement, wide range of applications, and cost- and time-effective multiplex for several parameters in different panels [23, 44, 45]. Several biomarkers can be cancer-specific since malignant cells of different histologic types can produce different patterns of proangiogenic factors, growth factors, and immune cells that are tumor related. The study of biomarker panels (Figure 3) and its spatial distribution (Figure 4) can be used for early diagnosis and assessment of therapy response [46]. This methodology can represent an ideal method to realize personalized therapies using efficient mIF panels and help to understand much better the cancer microenvironment, highlighting the benefit for exploring immune evasion mechanisms and finding potential biomarkers that allow researchers to assess the mechanism of action and predict and track response [47].
Microphotographs of representative examples of spatial-distribution visualization of different phenotypes analyzed. (A) distribution of individual cells using X and Y positions, (B) spatial localization of selected cells, and (C to F) distance measurements between malignant cells (MCs) and different cell populations.
The detection of multiple markers in the same tissue section can provide important and efficient means to apply this technology in disease diagnosis, prevention, and translational research. Multiplex immunoflourescence platforms have emerged more and more from translational research labs toward the clinic, increasing the opportunity to study and understand much better the tumor-immune interactions. This methodology and different image analysis strategies can give important information about immune cells’ coexpression and their spatial-pattern distribution in the tumor microenvironment. Development of multiplex immunoflourescence based-TSA system requires a very well-trained multidisciplinary team including pathologists, oncologists, and immunologists. In addition, this methodology requires automation to provide efficient and fast information as well as easy analysis methodologies for research pathologists that currently use this method.
The author would like to acknowledge the people that work in the laboratory of multiplex immunofluorescence, Mei Jang, Tong Li, Aerole Tanhemon, and Barbara Mino; the pathology team that work in the multiplex image analysis; as well as the chair of the Department of Translational Molecular Pathology, Dr. Ignacio Wistuba.
The author does not have any type of competing interest.
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