The hemagglutination assay and the hemagglutination inhibition assay were developed in 1941–42 by American virologist George Hirst as methods for quantifying the relative concentration of viruses, bacteria, or antibodies. HA and HI apply the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells bind to the hemagglutinin glycoprotein found on the surface of influenza virus and create a network, or lattice structure, of interconnected RBC’s and virus particles; the agglutinated lattice maintains the RBC’s in a suspended distribution viewed as a diffuse reddish solution. The formation of the lattice depends on the concentrations of the virus and RBC’s, when the relative virus concentration is too low, the RBC’s are not constrained by the lattice and settle to the bottom of the well. Hemagglutination is observed in the presence of staphylococci and other bacterial species, similar to the mechanism viruses use to cause agglutination of erythrocytes; the RBC’s used in HA and HI assays are from chickens, horses, guinea pigs, or humans depending on the selectivity of the targeted virus or bacterium and the associated surface receptors on the RBC.
A general procedure for HA is as follows, a serial dilution of virus is prepared across the rows in a U or V- bottom shaped 96-well microtiter plate. The most concentrated sample in the first well is diluted to be 1/5x of the stock, subsequent wells are two-fold dilutions; the final well serves as a negative control with no virus. Each row of the plate has a different virus and the same pattern of dilutions. After serial dilutions, a standardized concentration of RBCs is mixed gently; the plate is incubated for 30 minutes at room temperature. Following the incubation period, the assay can be analyzed to distinguish between agglutinated and non-agglutinated wells; the images across a row will progress from agglutinated wells with high virus concentration and a diffuse reddish appearance to a series of wells with low virus concentrations containing a dark red pellet, or button, in the center of the well. The low concentration wells appear nearly identical to the no-virus negative control well; the button appearance occurs because the RBC’s are not held in the agglutinated lattice structure and settle into the low point of the U or V-bottom well.
The transition from agglutinated to non-agglutinated wells occurs distinctively, within 1 to 2 wells. The relative concentration, or titer, of the virus sample is based on the well with the last agglutinated appearance before a pellet is observed. Relative to the initial viral stock concentration, the virus concentration in this well will be some dilution of the stock, for example, 1/40-fold; the titer value of that sample is the inverse of the dilution, i.e. 40. In some cases, the virus is so dilute that agglutinated wells are never observed. In that case, the titer of these samples is assigned as 5, indicating the highest possible concentration, but the accuracy of that value is low. Alternatively, if the relative concentration of the virus is high and the wells never transition to a button appearance; the titer value is commonly assigned to be the highest dilution, such as 5120. HI is related to the HA assay, but includes anti-viral antibodies as “inhibitors” to interfere with the virus-RBC interaction.
The goal is to characterize the concentration of antibodies in the antiserum or other samples containing antibodies. The HI assay is performed by creating a dilution series of antiserum across the rows of a 96-well microtiter plate; each row would be a different sample. A standardized amount of virus or bacteria is added to each well, the mixture is allowed to incubate at room temperature for 30 minutes; the last well in each row would be a negative control with no virus added. During the incubation, antibodies bind to the viral particles, if the concentration and binding affinity of the antibodies are high enough, the viral particles are blocked from causing hemagglutination. Next, a standardized amount of RBCs is added to each well and allowed to incubate at room temperature for an additional 30 minutes; the resulting HI plate images progress from non-agglutinated, “button” wells with high antibody concentration to agglutinated, red diffuse wells with low antibody concentration. The HI titer value is the inverse of the last dilution of serum that inhibited hemagglutination.
The preceding descriptions of the HA and HI processes are generalized, specific details can vary depending on the operator and laboratory. For example, serial dilutions across the rows is described, but some laboratories use an alternate orientation and perform dilutions down the columns instead; the starting dilution, serial dilution factor, incubation times, choice of U or V-bottom plate can depend on the specific laboratory. HA and HI have the advantages that the assays are simple, use inexpensive and available instruments and supplies, provide results within a few hours; the assays are well established in many laboratories around the world, allowing some measure of credibility and standardization. Optimal and reliable results require controlling several variables, such as incubation times, red blood cell concentration, type of red blood cell. Non-specific factors in the sample can lead to interference and incorrect titer values. For example, molecules in the sample other than virus-specific antibodies can inhibit agglutination between virus and RBC’s, as well as blocking ant
Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, is a protective response involving immune cells, blood vessels, molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, initiate tissue repair; the five classical signs of inflammation are heat, redness and loss of function. Inflammation is a generic response, therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, specific for each pathogen. Too little inflammation could lead to progressive tissue destruction by the harmful stimulus and compromise the survival of the organism. In contrast, chronic inflammation may lead to a host of diseases, such as hay fever, atherosclerosis, rheumatoid arthritis, cancer. Inflammation is therefore closely regulated by the body. Inflammation can be classified as either chronic.
Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Inflammation is not a synonym for infection. Infection describes the interaction between the action of microbial invasion and the reaction of the body's inflammatory response—the two components are considered together when discussing an infection, the word is used to imply a microbial invasive cause for the observed inflammatory reaction. Inflammation on the other hand describes purely the body's immunovascular response, whatever the cause may be.
But because of how the two are correlated, words ending in the suffix -itis are sometimes informally described as referring to infection. For example, the word urethritis means only "urethral inflammation", but clinical health care providers discuss urethritis as a urethral infection because urethral microbial invasion is the most common cause of urethritis, it is useful to differentiate inflammation and infection because there are typical situations in pathology and medical diagnosis where inflammation is not driven by microbial invasion – for example, trauma and autoimmune diseases including type III hypersensitivity. Conversely, there is pathology where microbial invasion does not cause the classic inflammatory response – for example, parasitosis or eosinophilia. Acute inflammation is a short-term process appearing within a few minutes or hours and begins to cease upon the removal of the injurious stimulus, it involves a coordinated and systemic mobilization response locally of various immune and neurological mediators of acute inflammation.
In a normal healthy response, it becomes activated, clears the pathogen and begins a repair process and ceases. It is characterized by five cardinal signs:An acronym that may be used to remember the key symptoms is "PRISH", for pain, immobility and heat; the traditional names for signs of inflammation come from Latin: Dolor Calor Rubor Tumor Functio laesa The first four were described by Celsus, while loss of function was added by Galen. However, the addition of this fifth sign has been ascribed to Thomas Sydenham and Virchow. Redness and heat are due to increased blood flow at body core temperature to the inflamed site. Loss of function has multiple causes. Acute inflammation of the lung does not cause pain unless the inflammation involves the parietal pleura, which does have pain-sensitive nerve endings; the process of acute inflammation is initiated by resident immune cells present in the involved tissue resident macrophages, dendritic cells, Kupffer cells and mast cells. These cells possess surface receptors known as pattern recognition receptors, which recognize two subclasses of molecules: pathogen-associated molecular patterns and damage-associated molecular patterns.
PAMPs are compounds that are associated with various pathogens, but which are distinguishable from host molecules. DAMPs are compounds that are associated with host-related cell damage. At the onset of an infection, burn, or other injuries, these cells undergo activation and release inflammatory mediators responsible for the clinical signs of inflammation. Vasodilation and its resulting increased blood flow causes increased heat. Increased permeability of the blood vessels results in an exudation of plasma proteins and fluid into the tissue, which manifests itself as swelling; some of the released mediators such as bradykinin increase the sensitivity to pain. The mediator molecules alter the blood vessels to
Red blood cell
Red blood cells known as RBCs, red cells, red blood corpuscles, erythroid cells or erythrocytes, are the most common type of blood cell and the vertebrate's principal means of delivering oxygen to the body tissues—via blood flow through the circulatory system. RBCs take up oxygen in the lungs, or gills of fish, release it into tissues while squeezing through the body's capillaries; the cytoplasm of erythrocytes is rich in hemoglobin, an iron-containing biomolecule that can bind oxygen and is responsible for the red color of the cells and the blood. The cell membrane is composed of proteins and lipids, this structure provides properties essential for physiological cell function such as deformability and stability while traversing the circulatory system and the capillary network. In humans, mature red blood cells are oval biconcave disks, they lack most organelles, in order to accommodate maximum space for hemoglobin. 2.4 million new erythrocytes are produced per second in human adults. The cells develop in the bone marrow and circulate for about 100–120 days in the body before their components are recycled by macrophages.
Each circulation takes about 60 seconds. A quarter of the cells in the human body are red blood cells. Nearly half of the blood's volume is red blood cells. Packed red blood cells are red blood cells that have been donated and stored in a blood bank for blood transfusion. All vertebrates, including all mammals and humans, have red blood cells. Red blood cells are cells present in blood; the only known vertebrates without red blood cells are the crocodile icefish. While they no longer use hemoglobin, remnants of hemoglobin genes can be found in their genome. Vertebrate red blood cells consist of hemoglobin, a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules in the lungs or gills and release them throughout the body. Oxygen can diffuse through the red blood cell's cell membrane. Hemoglobin in the red blood cells carries some of the waste product carbon dioxide back from the tissues. Myoglobin, a compound related to hemoglobin, acts to store oxygen in muscle cells.
The color of red blood cells is due to the heme group of hemoglobin. The blood plasma alone is straw-colored, but the red blood cells change color depending on the state of the hemoglobin: when combined with oxygen the resulting oxyhemoglobin is scarlet, when oxygen has been released the resulting deoxyhemoglobin is of a dark red burgundy color. However, blood can appear bluish when seen through skin. Pulse oximetry takes advantage of the hemoglobin color change to directly measure the arterial blood oxygen saturation using colorimetric techniques. Hemoglobin has a high affinity for carbon monoxide, forming carboxyhemoglobin, a bright red in color. Flushed, confused patients with a saturation reading of 100% on pulse oximetry are sometimes found to be suffering from carbon monoxide poisoning. Having oxygen-carrying proteins inside specialized cells was an important step in the evolution of vertebrates as it allows for less viscous blood, higher concentrations of oxygen, better diffusion of oxygen from the blood to the tissues.
The size of red blood cells varies among vertebrate species. The red blood cells of mammals are shaped as biconcave disks: flattened and depressed in the center, with a dumbbell-shaped cross section, a torus-shaped rim on the edge of the disk; this shape allows for a high surface-area-to-volume ratio to facilitate diffusion of gases. However, there are some exceptions concerning shape in the artiodactyl order, which displays a wide variety of bizarre red blood cell morphologies: small and ovaloid cells in llamas and camels, tiny spherical cells in mouse deer, cells which assume fusiform, lanceolate and irregularly polygonal and other angular forms in red deer and wapiti. Members of this order have evolved a mode of red blood cell development different from the mammalian norm. Overall, mammalian red blood cells are remarkably flexible and deformable so as to squeeze through tiny capillaries, as well as to maximize their apposing surface by assuming a cigar shape, where they efficiently release their oxygen load.
Red blood cells in mammals are unique amongst vertebrates. Red blood cells of mammals cells have nuclei during early phases of erythropoiesis, but extrude them during development as they mature; the red blood cells without nuclei, called reticulocytes, subsequently lose all other cellular organelles such as their mitochondria, Golgi apparatus and endoplasmic reticulum. The spleen acts as a reservoir of red blood cells. In some other mammals such as dogs and horses, the spl
A nephelometer is an instrument for measuring the concentration of suspended particulates in a liquid or gas colloid. A nephelometer measures suspended particulates by employing a light beam and a light detector set to one side of the source beam. Particle density is a function of the light reflected into the detector from the particles. To some extent, how much light reflects for a given density of particles is dependent upon properties of the particles such as their shape and reflectivity. Nephelometers are calibrated to a known particulate use environmental factors to compensate lighter or darker colored dusts accordingly. K-factor is determined by the user by running the nephelometer next to an air sampling pump and comparing results. There are a wide variety of research-grade nephelometers on the market as well as open source varieties; the main uses of nephelometers relate to air quality measurement for pollution monitoring, climate monitoring, visibility. Airborne particles are either biological contaminants, particulate contaminants, gaseous contaminants, or dust.
The chart to the left shows the sizes of various particulate contaminants. This information is helpful toward understanding the character of particulate pollution inside a building or in the ambient air, it is useful for understanding the cleanliness level in a controlled environment. Biological contaminants include mold, bacteria, animal dander, dust mites, human skin cells, cockroach parts, or anything alive or living at one time, they are the biggest enemy of indoor air quality specialists because they are contaminants that cause health problems. Levels of biological contamination depend on humidity and temperature that supports the livelihood of micro-organisms; the presence of pets, plants and insects will raise the level of biological contamination. Sheath air is clean filtered air that surrounds the aerosol stream to prevent particulates from circulating or depositing within the optic chamber. Sheath air prevents contamination caused by build-up and deposits, improves response time by containing the sample, improves maintenance by keeping the optic chamber clean.
The nephelometer creates the sheath air by passing air through a zero filter before beginning the sample. Nephelometers are used in global warming studies measuring the global radiation balance. Three wavelength nephelometers fitted with a backscatter shutter can determine the amount of solar radiation, reflected back into space through dust and particulate matter; this reflected light influences the amount of radiation reaching the earth's lower atmosphere and warming the planet. Nephelometers are used for measurement of visibility with simple one-wavelength nephelometers used throughout the world by many EPAs. Nephelometers, through the measurement of light scattering, can determine visibility in distance through the application of a conversion factor called Koschmieder's formula. In medicine, nephelometry is used to measure immune function. Gas-phase nephelometers are used in the detection of smoke and other particles of combustion. In such use, the apparatus is referred to as an aspirated smoke detector.
These have the capability to detect low particle concentrations and are therefore suitable to protecting sensitive or valuable electronic equipment, such as mainframe computers and telephone switches. Because optical properties depend on suspended particle size, a stable synthetic material called "Formazin" with uniform particle size is used as a standard for calibration and reproducibility; the unit is called Formazin Turbidity Unit. Nephelometric Turbidity Units specified by United States Environmental Protection Agency is a special case of FTU, where a white light source and certain geometrical properties of the measurement apparatus are specified. Formazin Nephelometric Units, prescribed for 9 measurements of turbidity in water treatment by ISO 7027, another special case of FTU with near infrared light and 90° scatter. Formazin Attenuation Units specified by ISO 7027 for water treatment standards for turbidity measurements at 0° a special case of FTU. Formazin Backscatter Units, not part of a standard, is the unit of optical backscatter detectors, measured at c.
180° a special case of FTU. European Brewery Convention turbidity units Concentration Units Optical Density Jackson "Candle" Turbidity Units Helms Units American Society of Brewing Chemists turbidity units Parts Per Million of standard substance, such as PPM/DE "Trübungseinheit/Formazin" a German standard, now replaced by the FNU unit. Diatomaceous earth an older standard, now obsoleteA more popular term for this instrument in water quality testing is a turbidimeter. However, there can be differences between models of turbidimeters, depending upon the arrangement of the source beam and the detector. A nephelometric turbidimeter always monitors light reflected off the particles and not attenuation due to cloudiness. In the United States environmental monitoring the turbidity standard unit is called Nephelometric Turbidity Units, while the international standard unit is called Formazin Nephelometric Unit; the most applicable unit is Formazin Turbidity Unit, although different measurement methods can give quite different values as reported in FTU.
Gas-phase nephelometers are used to study the atmosphere. These can provide information on atmospheric albedo. ISO 7027 Water purification
Ouchterlony double immunodiffusion
Ouchterlony double immunodiffusion is an immunological technique used in the detection and quantification of antibodies and antigens, such as immunoglobulins and extractable nuclear antigens. The technique is named after Örjan Ouchterlony, the Swedish physician who invented the test in 1948. A gel plate is cut to form a series of holes in the gel. A sample extract of interest is placed in one well, sera or purified antibodies are placed in another well and the plate left for 48 hours to develop. During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells. Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form an immune complex; the immune complex precipitates in the gel to give a thin white line, a visual signature of antigen recognition. The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple wells with different antibodies or mixtures of antibodies, antigen-antibody reactivity can be seen by observing between which wells the precipitate is observed.
When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected. The zone of equivalence lines may give partial identity, or a non-identity. Precipitation occurs with most antigens. Antibodies have at least two antigen binding sites, thus large aggregates or gel-like lattices of antigen and antibody are formed. Experimentally, an increasing amount of antigen is added to a constant amount of antibody in solution. At low antigen concentration, all of the antibody is contained in the precipitate; this is called the antibody-excess zone. As more antigen is added, the amount of protein precipitated increases until the antigen/antibody molecules are at an optimal ratio; this is known as the zone of equivalence point. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease; this is known as the antigen excess zone. Bailey, Graham S.. "135: Ouchterlony Double Immunodiffusion". In Walker, John M.
The Protein Protocols Handbook. VII: Immunochemical Techniques. Totowa, New Jersey: Humana Press. Pp. 749–752. Doi:10.1007/978-1-60327-259-9_135. Retrieved 2012-11-22. Ouchterlony, O.. "In vitro method for testing the toxin-producing capacity of diphtheria bacteria". Acta pathologica et microbiologica Scandinavica. 25: 186–191. Doi:10.1111/j.1699-0463.1948.tb00655.x. PMID 18887479. Ouchterlony, Örjan. "Antigen-antibody reactions in gels". Acta pathologica et microbiologica Scandinavica. 26: 507–515. Doi:10.1111/j.1699-0463.1949.tb00751.x. PMID 18143039. Ouchterlony, O.. "Diffusion-in-gel methods for immunological analysis". Progress in allergy. 5: 1–78. PMID 13578996. Ouchterlony, O.. "Diffusion-in-gel methods for immunological analysis. II". Progress in allergy. 6: 30–154. PMID 14482809. Ouchterlony, O.. A.. Weir, Daniel Mackay, ed. Handbook of Experimental Immunology. 1. Immunochemistry. Oxford, England: Blackwell Scientific. Pp. 32.1–32.50. Retrieved 2012-11-23. At Google Books "Ouchterlony Analysis". Medical Immunology 544.
University of California, Irvine College of Medicine. Fall 2011. Archived from the original on 2012-11-23. Retrieved 2012-11-23. "Ouchterlony Double Diffusion - Patterns: Theory". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. Archived from the original on 2012-11-23. Retrieved 2012-12-24. "Ouchterlony Double Diffusion - Patterns: Procedure". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. Archived from the original on 2012-12-24. Retrieved 2012-12-24. "Ouchterlony Double Diffusion - Titration: Theory". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. Archived from the original on 2012-11-23. Retrieved 2012-11-23. "Ouchterlony Double Diffusion - Titration: Procedure". Value @ Amrita. India: Amrita Vishwa Vidyapeetham University. 2012. Archived from the original on 2012-12-24. Retrieved 2012-12-24. "BSCI423: Lab 5. Precipitation". College of Computer and Natural Sciences, University of Maryland, College Park. Archived from the original on 2012-11-23. Retrieved 2012-11-23.
Simulator at University of California, Irvine "Ouchterlony double immunodiffusion". Retrieved 2017-05-15. Photograph of Ouchterlony double immunodiffusion plate with unstained precipitin lines of full identity and non-identity. "Diffusion Patterns". Immunodiffusion principles and application. Retrieved 2017-05-19. Photographs of Ouchterlony immunodiffusion patterns showing stained precipitin lines of full identity, partial identity and non-identity
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins known as antibodies, reacting with the proteins to be separated or characterized; the methods were used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis, crossed immunoelectrophoresis, rocket-immunoelectrophoresis, fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen. Agarose as 1% gel slabs of about 1 mm thickness buffered at high pH is traditionally preferred for the electrophoresis as well as the reaction with antibodies; the agarose was chosen as the gel matrix because it has large pores allowing free passage and separation of proteins, but provides an anchor for the immunoprecipitates of protein and specific antibodies.
The high pH was chosen because antibodies are immobile at high pH. An electrophoresis equipment with a horizontal cooling plate was recommended for the electrophoresis. Immunoprecipitates may be seen in the wet agarose gel, but are stained with protein stains like Coomassie Brilliant Blue in the dried gel. In contrast to SDS-gel electrophoresis, the electrophoresis in agarose allows native conditions, preserving the native structure and activities of the proteins under investigation, therefore immunoelectrophoresis allows characterization of enzyme activities and ligand binding etc. in addition to electrophoretic separation. The immunoelectrophoretic analysis ad modum Grabar is the classical method of immunoelectrophoresis. Proteins are separated by electrophoresis antibodies are applied in a trough next to the separated proteins and immunoprecipitates are formed after a period of diffusion of the separated proteins and antibodies against each other; the introduction of the immunoelectrophoretic analysis gave a great boost to protein chemistry, some of the first results were the resolution of proteins in biological fluids and biological extracts.
Among the important observations made were the great number of different proteins in serum, the existence of several immunoglobulin classes and their electrophoretic heterogeneity. Crossed immunoelectrophoresis is called two-dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad modum Laurell. In this method the proteins are first separated during the first dimension electrophoresis instead of the diffusion towards the antibodies, the proteins are electrophoresed into an antibody-containing gel in the second dimension. Immunoprecipitation will take place during the second dimension electrophorsis and the immunoprecipitates have a characteristic bell-shape, each precipitate representing one antigen, the position of the precipitate being dependent on the amount of protein as well as the amount of specific antibody in the gel, so relative quantification can be performed; the sensitivity and resolving power of crossed immunoelectrophoresis is than that of the classical immunoelectrophoretic analysis and there are multiple variations of the technique useful for various purposes.
Crossed immunoelectrophoresis has been used for studies of proteins in biological fluids human serum, biological extracts. Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis; the method has been used for quantitation of human serum proteins before automated methods became available. Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative immunoelectrophorsis used for detailed measurement of proteins in fractions from protein separation experiments. Affinity immunoelectrophoresis is based on changes in the electrophoretic pattern of proteins through specific interaction or complex formation with other macromolecules or ligands. Affinity immunoelectrophoresis has been used for estimation of binding constants, as for instance with lectins or for characterization of proteins with specific features like glycan content or ligand binding; some variants of affinity immunoelectrophoresis are similar to affinity chromatography by use of immobilized ligands.
The open structure of the immunoprecipitate in the agarose gel will allow additional binding of radioactively labeled antibodies to reveal specific proteins. This variation has been used for identification of allergens through reaction with IgE. Two factors determine that immunoelectrophoretic methods are not used. First they are rather require some manual expertise. Second they require rather large amounts of polyclonal antibodies. Today gel electrophoresis followed by electroblotting is the preferred method for protein characterization because its ease of operation, its high sensitivity, its low requirement for specific antibodies. In addition proteins are separated by gel electrophoresis on the basis of their apparent molecular weight, not accomplished by immunoelectrophoresis, but immunoelectrophoretic methods are still useful when non-reducing conditions are needed. Comprehensive text edited by Niels H. Axelsen in Scandinavian Journal of Immunology, 1975 Volume 4 Supplement Immunoelectrophoresis at the US National Library of Medicine Medical Subject Headings http://www.lib.mcg.edu/edu/esimmuno/ch4/immelec.htm Immuno-Electrophoresis.
A lymphocyte is one of the subtypes of a white blood cell in a vertebrate's immune system. Lymphocytes include natural killer cells, T cells, B cells, they are the main type of cell found in lymph, which prompted the name "lymphocyte". The three major types of lymphocyte are B cells and natural killer cells. Lymphocytes can be identified by their large nucleus. T cells and B cells are the major cellular components of the adaptive immune response. T cells are involved in cell-mediated immunity, whereas B cells are responsible for humoral immunity; the function of T cells and B cells is to recognize specific "non-self" antigens, during a process known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells. B cells respond to pathogens by producing large quantities of antibodies which neutralize foreign objects like bacteria and viruses. In response to pathogens some T cells, called T helper cells, produce cytokines that direct the immune response, while other T cells, called cytotoxic T cells, produce toxic granules that contain powerful enzymes which induce the death of pathogen-infected cells.
Following activation, B cells and T cells leave a lasting legacy of the antigens they have encountered, in the form of memory cells. Throughout the lifetime of an animal, these memory cells will "remember" each specific pathogen encountered, are able to mount a strong and rapid response if the same pathogen is detected again. NK cells are a part of the innate immune system and play a major role in defending the host from tumors and virally infected cells. NK cells distinguish infected cells and tumors from normal and uninfected cells by recognizing changes of a surface molecule called MHC class I. NK cells are activated in response to a family of cytokines called interferons. Activated NK cells release cytotoxic granules which destroy the altered cells, they are named "natural killer cells" because they do not require prior activation in order to kill cells which are missing MHC class I. Mammalian stem cells differentiate into several kinds of blood cell within the bone marrow; this process is called haematopoiesis.
All lymphocytes originate, during this process, from a common lymphoid progenitor before differentiating into their distinct lymphocyte types. The differentiation of lymphocytes follows various pathways in a hierarchical fashion as well as in a more plastic fashion; the formation of lymphocytes is known as lymphopoiesis. B cells mature into B lymphocytes in the bursa equivalent, which in humans is the GALT, thought to be located in the Peyer's patches of the intestine, while T cells migrate to and mature in a distinct organ, called the thymus. Following maturation, the lymphocytes enter the circulation and peripheral lymphoid organs where they survey for invading pathogens and/or tumor cells; the lymphocytes involved in adaptive immunity differentiate further after exposure to an antigen. Effector lymphocytes function to eliminate the antigen, either by releasing antibodies, cytotoxic granules or by signaling to other cells of the immune system. Memory T cells remain in the peripheral tissues and circulation for an extended time ready to respond to the same antigen upon future exposure.
Microscopically, in a Wright's stained peripheral blood smear, a normal lymphocyte has a large, dark-staining nucleus with little to no eosinophilic cytoplasm. In normal situations, the coarse, dense nucleus of a lymphocyte is the size of a red blood cell; some lymphocytes show a clear perinuclear zone around the nucleus or could exhibit a small clear zone to one side of the nucleus. Polyribosomes are a prominent feature in the lymphocytes and can be viewed with an electron microscope; the ribosomes are involved in protein synthesis, allowing the generation of large quantities of cytokines and immunoglobulins by these cells. It is impossible to distinguish between B cells in a peripheral blood smear. Flow cytometry testing is used for specific lymphocyte population counts; this can be used to determine the percentage of lymphocytes that contain a particular combination of specific cell surface proteins, such as immunoglobulins or cluster of differentiation markers or that produce particular proteins.
In order to study the function of a lymphocyte by virtue of the proteins it generates, other scientific techniques like the ELISPOT or secretion assay techniques can be used. In the circulatory system, they move from lymph node to lymph node; this contrasts with macrophages. A lymphocyte count is part of a peripheral complete blood cell count and is expressed as the percentage of lymphocytes to the total number of white blood cells counted. A general increase in the number of lymphocytes is known as lymphocytosis, whereas a decrease is known as lymphocytopenia. An increase in lymphocyte concentration is a sign of a viral infection. A high lymphocyte count wi