{"id":860,"date":"2016-11-04T03:35:33","date_gmt":"2016-11-04T03:35:33","guid":{"rendered":"https:\/\/courses.lumenlearning.com\/microbiology\/?post_type=chapter&#038;p=860"},"modified":"2016-11-11T00:37:34","modified_gmt":"2016-11-11T00:37:34","slug":"agglutination-assays","status":"publish","type":"chapter","link":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/chapter\/agglutination-assays\/","title":{"raw":"Agglutination Assays","rendered":"Agglutination Assays"},"content":{"raw":"<div class=\"textbox learning-objectives\">\r\n<h3>Learning Objectives<\/h3>\r\n<ul>\r\n \t<li>Compare direct and indirect agglutination<\/li>\r\n \t<li>Identify various uses of hemagglutination in the diagnosis of disease<\/li>\r\n \t<li>Explain how blood types are determined<\/li>\r\n \t<li>Explain the steps used to cross-match blood to be used in a transfusion<\/li>\r\n<\/ul>\r\n<\/div>\r\nIn addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called <strong>agglutination<\/strong> (<a href=\".\/chapter\/overview-of-specific-adaptive-immunity\/\" target=\"_blank\">Figure\u00a07 in\u00a0Overview of Specific Adaptive Immunity<\/a>). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or <strong>microtiter plate<\/strong> (Figure\u00a01). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.\r\n\r\n[caption id=\"\" align=\"aligncenter\" width=\"651\"]<img class=\"\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172929\/OSC_Microbio_20_03_Microt.jpg\" alt=\"A plastic plate about the size of a hand with wells in a grid. The top is labeled 1-12 and the side is labeled A-H.\" width=\"651\" height=\"488\" data-media-type=\"image\/jpeg\" \/> Figure\u00a01. Microtiter plates are used for conducting numerous reactions simultaneously in an array of wells. (credit: modification of work by \"Microrao\"\/Wikimedia)[\/caption]\r\n<h2>Agglutination of Bacteria and Viruses<\/h2>\r\nThe use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca <strong>Lancefield<\/strong> working with her colleagues A.R. <strong>Dochez<\/strong> and Oswald <strong>Avery<\/strong>.[footnote]Lancefield, Rebecca C., \"The Antigenic Complex of <em>Streptococcus haemoliticus<\/em>. I. Demonstration of a Type-Specific Substance in Extracts of <em>Streptococcus haemolyticus<\/em>,\" <em>The Journal of Experimental Medicine<\/em> 47, no. 1 (1928): 91-103.[\/footnote] She used antibodies to identify <strong>M protein<\/strong>, a virulence factor on streptococci that is necessary for the bacteria\u2019s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.\r\n\r\nLancefield used antisera to show that different strains of the same species of <strong>streptococci<\/strong> express different versions of M protein, which explains why children can come down with <strong>strep throat<\/strong> repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called <strong>serovars<\/strong> because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.\r\n\r\n[caption id=\"attachment_1735\" align=\"alignright\" width=\"450\"]<img class=\" wp-image-1735\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001546\/OSC_Microbio_20_03_Lance1.jpg\" alt=\"Photo of 6 wells. Well 4 has blue spots. All the other wells have a clear blue color.\" width=\"450\" height=\"289\" \/> Figure\u00a02. Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology)[\/caption]\r\n\r\nThe method developed by Lancefield is a <strong>direct agglutination assay<\/strong>, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert <strong>latex beads<\/strong>. This technique is called an <strong>indirect agglutination assay<\/strong> (or <strong>latex fixation assay<\/strong>), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure\u00a02). Indirect assays can be used to detect the presence of either antibodies or specific antigens.\r\n\r\nTo identify antibodies in a patient\u2019s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure\u00a03). This technique is most often used when looking for <strong>IgM<\/strong> antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for <strong>rheumatoid factor (RF)<\/strong> to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient\u2019s own <strong>IgG<\/strong>. RF will agglutinate IgG-coated latex beads.\r\n\r\nIn the reverse test, soluble antigens can be detected in a patient\u2019s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure\u00a03).\r\n\r\n[caption id=\"\" align=\"aligncenter\" width=\"1200\"]<img src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172935\/OSC_Microbio_20_03_Latex.jpg\" alt=\"(a) A diagram of positive agglutination test for antibodies. Large circles (latex beads) have smaller circles (antigens) on their surface. IgM antibodies (6 Y shapes attached at their base) are bound to the antigens. B) A diagram of positive agglutination test for antigens. The latex beads have antigens on them. Smaller blue ovals (pathogens) have circles (antigens) on their surface. The antigens bind to the antibodies.\" width=\"1200\" height=\"670\" data-media-type=\"image\/jpeg\" \/> Figure\u00a03. (a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. (b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies.[\/caption]\r\n\r\nAgglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the <strong>Widal test<\/strong>, used for the diagnosis of <strong>typhoid fever<\/strong>, looks for agglutination of <strong><em>Salmonella enterica<\/em> subspecies <em>typhi<\/em><\/strong> in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of <em>Salmonella<\/em>, as well as false negatives in patients with hyperproteinemia or immune deficiencies.\r\n\r\nIn addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone <strong>seroconversion<\/strong> when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.\r\n\r\nSimilar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient\u2019s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the <strong>titer<\/strong> during the <strong>acute phase<\/strong> of an infection versus the titer from the <strong>convalescent phase<\/strong> will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient\u2019s immune system is responding to the pathogen.\r\n<div class=\"textbox\">\r\n\r\nWatch this video that demonstrates agglutination reactions with latex beads.\r\n\r\nhttps:\/\/youtu.be\/oXL1rH11MTg\r\n\r\n<\/div>\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Think about It<\/h3>\r\n<ul>\r\n \t<li>How is agglutination used to distinguish serovars from each other?<\/li>\r\n \t<li>In a latex bead assay to test for antibodies in a patient's serum, with what are the beads coated?<\/li>\r\n \t<li>What has happened when a patient has undergone seroconversion?<\/li>\r\n<\/ul>\r\n<\/div>\r\n<h3>Hemagglutination<\/h3>\r\nAgglutination of red blood cells is called <strong>hemagglutination<\/strong>. One common assay that uses hemagglutination is the <strong>direct Coombs\u2019 test<\/strong>, also called the <strong>direct antihuman globulin test (DAT)<\/strong>, which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.\r\n\r\nThe Coombs\u2019 test is often employed when a newborn has <strong>jaundice<\/strong>, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs\u2019 test is used to determine whether the child\u2019s red blood cells have been bound by the mother\u2019s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs\u2019 tests include <strong>hemolytic transfusion reactions<\/strong>, <strong>autoimmune hemolytic anemia<\/strong>, infectious mononucleosis (caused by <strong>Epstein-Barr virus<\/strong>), <strong>syphilis<\/strong>, and <strong><em>Mycoplasma<\/em> pneumonia<\/strong>. A positive direct Coombs\u2019 test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).\r\n\r\nThe antibodies bound to red blood cells in these conditions are most often <strong>IgG<\/strong>, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding <strong>Coombs\u2019 reagent<\/strong>, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure\u00a04). The Coombs\u2019 reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.\r\n\r\nThere is also an <strong>indirect Coombs\u2019 test<\/strong> known as the <strong>indirect antiglobulin test (IAT)<\/strong>. This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum (Figure\u00a04). IAT can be used to screen pregnant women for antibodies that may cause <strong>hemolytic disease of the newborn<\/strong>. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.\r\n\r\n[caption id=\"attachment_1736\" align=\"aligncenter\" width=\"600\"]<a href=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001803\/OSC_Microbio_20_03_Coombs1.jpg\" target=\"_blank\"><img class=\"wp-image-1736\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001803\/OSC_Microbio_20_03_Coombs1-868x1024.jpg\" alt=\"A) Diagram of direct Coomb\u2019s test. 1. Blood sample from a patient with hemolytic anemia with antibodies attached to red blood cells. 2. Antibodies coat the patient\u2019s red blood cells. 3. A few drops of Coomb\u2019s reagent (containing anti-human antibodies) is mixed in with the patient\u2019s blood sample. 4. Agglutination reaction (clumping) is visible after cross-linkage of antibodies. B) Diagram of indirect Coomb\u2019s test. 1. Patient's serum containing antibodies is drawn. 2. Donor blood is added. 3. Patient's antibodies bind to donor's red blood cells. 4. A few drops of Coombs\u2019 reagent are mixed with the sample. 5. Agglutination reaction is observed.\" width=\"600\" height=\"708\" \/><\/a> Figure\u00a04. Click for a larger image. The steps in direct and indirect Coombs\u2019 tests are shown in the illustration.[\/caption]\r\n\r\nAntibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, <strong>influenza viruses<\/strong> have two different types of viral spikes called <strong>neuraminidase<\/strong> (N) and <strong>hemagglutinin<\/strong> (H), the latter named for its ability to agglutinate red blood cells (see <a href=\".\/chapter\/viruses\/\" target=\"_blank\">Viruses<\/a>). Thus, we can use red blood cells to detect the presence of influenza virus by <strong>direct hemagglutination assays<\/strong> (HA), in which the virus causes visible agglutination of red blood cells. The <strong>mumps<\/strong> and <strong>rubella<\/strong> viruses can also be detected using HA.\r\n\r\nMost frequently, a <strong>serial dilution<\/strong> viral agglutination assay is used to measure the <strong>titer<\/strong> or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure\u00a05).\r\n\r\n[caption id=\"\" align=\"aligncenter\" width=\"500\"]<img class=\"\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172945\/OSC_Microbio_20_03_HA.jpg\" alt=\"A photo of nonagglutination shows a red stripe. A diagram shows that without virus the cells from a compact pellet at the bottom of the well. A photo of agglutination shows a red spot and pink surrounding area. A diagram shows that the red blood cells and viruses form a clump.\" width=\"500\" height=\"447\" data-media-type=\"image\/jpeg\" \/> Figure\u00a05. A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology)[\/caption]\r\n\r\nA modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient\u2019s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a <strong>viral hemagglutination inhibition assay<\/strong> (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient\u2019s serum is the highest dilution that blocks agglutination (Figure\u00a06).\r\n\r\n[caption id=\"attachment_1737\" align=\"aligncenter\" width=\"500\"]<img class=\" wp-image-1737\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11002047\/OSC_Microbio_20_03_HIA1.jpg\" alt=\"A drawing of a well plate. The label at the top reads: Dilution. The rows are labeled: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. The columns are labeled: Sample A, titer = 128 (this spans columns 1 and 2). Sample B, no neutralizing antibody (spans columns 3 and 4). Sample C, titer = 64 spans columns 5 and 6.). Columns 1 and 2 have red dots in all rows but the bottom one. Columns 3 and 4 have no red dots. Columns 5 and 6 have red dots in all rows but the bottom two.\" width=\"500\" height=\"629\" \/> Figure\u00a06. In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest concentration at which agglutination occurs is the titer of the antibodies in the patient\u2019s serum. In the case of this test, Sample A shows a titer of 128, and Sample C shows a titer of 64. (credit: modification of work by Evan Burkala)[\/caption]\r\n\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Think about It<\/h3>\r\n<ul>\r\n \t<li>What is the mechanism by which viruses are detected in a hemagglutination assay?<\/li>\r\n \t<li>Which hemagglutination result tells us the titer of virus in a sample?<\/li>\r\n<\/ul>\r\n<\/div>\r\n<div class=\"textbox shaded\">\r\n<h3>Animals in the Laboratory<\/h3>\r\nMuch of what we know today about the human immune system has been learned through research conducted using animals\u2014primarily, mammals\u2014as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.\r\n\r\nConsider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.\r\n\r\nWe also use animals for the study of disease. The only way to grow <em>Treponema pallidum<\/em> for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch\u2019s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.\r\n\r\nWhile the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one\u2014indeed, one of Charles Darwin's daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document \"Guide for the Care and Use of Laboratory Animals\" makes it clear that the use of animals in research is a privilege granted by society to researchers.\r\n\r\nThe NIH guidelines are based on the principle of the three R\u2019s: replace, refine, and reduce. Researchers should strive to <em>replace<\/em> animal models with nonliving models, <em>replace<\/em> vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should <em>refine<\/em> husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that <em>reduce<\/em> the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.\r\n\r\nAt the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.\r\n\r\n<\/div>\r\n<div class=\"textbox\">Visit this site to <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK54050\/\" target=\"_blank\">view the NIH Guide for the Care and Use of Laboratory Animals<\/a>.<\/div>\r\n<h2>Blood Typing and Cross-Matching<\/h2>\r\nIn addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against <strong>blood types<\/strong> other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the <strong>ABO<\/strong> and <strong>Rh blood groups<\/strong> (see <a href=\".\/chapter\/hypersensitivities\/\" target=\"_blank\">Figure\u00a03 in\u00a0Hypersensitivities<\/a>).\r\n\r\nWhen units of blood are being considered for <strong>transfusion<\/strong>, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient\u2019s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure\u00a07, if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative.\r\n\r\nFollowing determination of the blood type, immediately prior to releasing the blood for transfusion, a <strong>cross-match<\/strong> is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, <strong>hemagglutination<\/strong> will occur. To confirm any negative test results and check for sensitized red blood cells, <strong>Coombs\u2019 reagent<\/strong> may be added to the mix to facilitate visualization of the antibody-red blood cell interaction.\r\n\r\nUnder some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation.\r\n\r\nRed blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, women who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an <strong>antibody screen<\/strong> test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit.\r\n\r\n[caption id=\"\" align=\"aligncenter\" width=\"1097\"]<img src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172951\/OSC_Microbio_20_03_BloodT.jpg\" alt=\"Three wells the first well (labeled anti-A) shows black spots and is labeled clumping. This is due to the agglutinated RBCs. The second well (labeled anti-B) looks smooth and has no clumping. The third well (labeled) anti-Rh shows clumping because it has spots.\" width=\"1097\" height=\"350\" data-media-type=\"image\/jpeg\" \/> Figure\u00a07. This sample of a commercially produced \"bedside\" card enables quick typing of both a recipient\u2019s and donor\u2019s blood before transfusion. The card contains three reaction sites or wells. One is coated with an anti-A antibody, one with an anti-B antibody, and one with an anti-Rh antibody. Agglutination of red blood cells in a given site indicates a positive identification of the blood antigens: in this case, A and Rh antigens for blood type A-positive.[\/caption]\r\n\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Think about It<\/h3>\r\n<ul>\r\n \t<li>If a patient's blood agglutinates with anti-B serum, what is the patient\u2019s blood type?<\/li>\r\n \t<li>What is a cross-match assay, and why is it performed?<\/li>\r\n<\/ul>\r\n<\/div>\r\nTable 1 summarizes the various kinds of agglutination assays discussed in this section.\r\n<table id=\"fs-id1167661289772\" class=\"span-all\" summary=\"Table titled: Mechanisms of Select Antibody-Antigen Assays. Type of Assay: Agglutination. Mechanism: Direct: Antibody is used to clump bacterial cells or other large structures. Example: Serotyping bacteria. Mechanism: Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum. Example: Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum. Type of assay: Hemagglutination. Mechanism: Direct: Some bacteria and viruses cross-link red blood cells and clump them together. Example: Diagnosing influenza, mumps, and measles. Mechanism: Direct Coombs\u2019 test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells in vivo. Example: Checking for maternal antibodies binding to neonatal red blood cells. Mechanism: Indirect Coombs\u2019 test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum in vitro. Example: Performing pretransfusion blood testing. Mechanism: Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination. Example: Diagnosing various viral diseases by the presence of patient antibodies against the virus. Mechanism: Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood. Example: Matches donor blood to recipient immune requirements.\">\r\n<thead>\r\n<tr>\r\n<th colspan=\"3\" data-align=\"center\">Table 1. Mechanisms of Select Antibody-Antigen Assays<\/th>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<th data-valign=\"top\" data-align=\"left\">Type of Assay<\/th>\r\n<th data-valign=\"top\" data-align=\"left\">Mechanism<\/th>\r\n<th data-valign=\"top\" data-align=\"left\">Example<\/th>\r\n<\/tr>\r\n<\/thead>\r\n<tbody>\r\n<tr valign=\"top\">\r\n<td rowspan=\"2\" data-valign=\"top\" data-align=\"left\">Agglutination<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Direct: Antibody is used to clump bacterial cells or other large structures<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Serotyping bacteria<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td data-valign=\"top\" data-align=\"left\">Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td rowspan=\"5\" data-valign=\"top\" data-align=\"left\">Hemagglutination<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Direct: Some bacteria and viruses cross-link red blood cells and clump them together<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Diagnosing influenza, mumps, and measles<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td data-valign=\"top\" data-align=\"left\">Direct Coombs\u2019 test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells <em>in vivo<\/em><\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Checking for maternal antibodies binding to neonatal red blood cells<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td data-valign=\"top\" data-align=\"left\">Indirect Coombs\u2019 test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum <em>in vitro<\/em><\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Performing pretransfusion blood testing<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td data-valign=\"top\" data-align=\"left\">Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Diagnosing various viral diseases by the presence of patient antibodies against the virus<\/td>\r\n<\/tr>\r\n<tr valign=\"top\">\r\n<td data-valign=\"top\" data-align=\"left\">Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood<\/td>\r\n<td data-valign=\"top\" data-align=\"left\">Matches donor blood to recipient immune requirements<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Key Concepts and Summary<\/h3>\r\n<ul>\r\n \t<li>Antibodies can agglutinate cells or large particles into a visible matrix. <strong>Agglutination<\/strong> tests are often done on cards or in <strong>microtiter plates<\/strong> that allow multiple reactions to take place side by side using small volumes of reagents.<\/li>\r\n \t<li>Using antisera against certain proteins allows identification of <strong>serovars<\/strong> within species of bacteria.<\/li>\r\n \t<li>Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through <strong>seroconversion<\/strong> and the level of antibodies becomes detectable.<\/li>\r\n \t<li>Agglutination of latex beads in <strong>indirect agglutination assays<\/strong> can be used to detect the presence of specific antigens or specific antibodies in patient serum.<\/li>\r\n \t<li>The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct <strong>Coombs\u2019 test<\/strong>, which uses Coombs\u2019 reagent to cross-link antibodies bound to red blood cells and facilitate <strong>hemagglutination<\/strong>.<\/li>\r\n \t<li>Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the <strong>direct hemagglutination assay<\/strong>, most often used to determine the titer of virus in solution.<\/li>\r\n \t<li><strong>Neutralization assays<\/strong> quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.<\/li>\r\n \t<li>Hemagglutination assays are also used to screen and <strong>cross-match<\/strong> donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<div class=\"textbox exercises\">\r\n<h3>Multiple Choice<\/h3>\r\nWe use antisera to distinguish between various ________ within a species of bacteria.\r\n<ol style=\"list-style-type: lower-alpha;\">\r\n \t<li>isotypes<\/li>\r\n \t<li>serovars<\/li>\r\n \t<li>subspecies<\/li>\r\n \t<li>lines<\/li>\r\n<\/ol>\r\n[reveal-answer q=\"443301\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"443301\"]Answer b.\u00a0We use antisera to distinguish between various serovars within a species of bacteria.[\/hidden-answer]\r\n\r\nWhen using antisera to characterize bacteria, we will often link the antibodies to ________ to better visualize the agglutination.\r\n<ol style=\"list-style-type: lower-alpha;\">\r\n \t<li>latex beads<\/li>\r\n \t<li>red blood cells<\/li>\r\n \t<li>other bacteria<\/li>\r\n \t<li>white blood cells<\/li>\r\n<\/ol>\r\n[reveal-answer q=\"71926\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"71926\"]Answer a.\u00a0When using antisera to characterize bacteria, we will often link the antibodies to latex beads to better visualize the agglutination.[\/hidden-answer]\r\n\r\nThe antibody screening test that is done along with pretransfusion blood typing is used to ensure that the recipient\r\n<ol style=\"list-style-type: lower-alpha;\">\r\n \t<li>does not have a previously undetected bacterial or viral infection.<\/li>\r\n \t<li>is not immunocompromised.<\/li>\r\n \t<li>actually does have the blood type stated in the online chart.<\/li>\r\n \t<li>is not making antibodies against antigens outside the ABO or Rh systems.<\/li>\r\n<\/ol>\r\n[reveal-answer q=\"92382\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"92382\"]Answer d. This test is done to ensure\u00a0that the recipient is not making antibodies against antigens outside the ABO or Rh systems.[\/hidden-answer]\r\n\r\nThe direct Coombs\u2019 test is designed to detect when people have a disease that causes them to\r\n<ol style=\"list-style-type: lower-alpha;\">\r\n \t<li>have an excessively high fever.<\/li>\r\n \t<li>quit making antibodies.<\/li>\r\n \t<li>make too many red blood cells.<\/li>\r\n \t<li>produce antibodies that bind to their own red blood cells.<\/li>\r\n<\/ol>\r\n[reveal-answer q=\"511979\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"511979\"]Answer d. The direct Coombs\u2019 test is designed to detect when people have a disease that causes them to\u00a0produce antibodies that bind to their own red blood cells.[\/hidden-answer]\r\n\r\nViral hemagglutination assays only work with certain types of viruses because\r\n<ol style=\"list-style-type: lower-alpha;\">\r\n \t<li>the virus must be able to cross-link red blood cells directly.<\/li>\r\n \t<li>the virus must be able to lyse red blood cells.<\/li>\r\n \t<li>the virus must not be able to lyse red blood cells.<\/li>\r\n \t<li>other viruses are too dangerous to work with in a clinical lab setting.<\/li>\r\n<\/ol>\r\n[reveal-answer q=\"862143\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"862143\"]Answer a.\u00a0Viral hemagglutination assays only work with certain types of viruses because\u00a0the virus must be able to cross-link red blood cells directly.[\/hidden-answer]\r\n\r\n<\/div>\r\n<div class=\"textbox exercises\">\r\n<h3>Fill in the Blank<\/h3>\r\nIn the major cross-match, we mix ________ with the donor red blood cells and look for agglutination.\r\n[reveal-answer q=\"903843\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"903843\"]In the major cross-match, we mix <strong>patient serum<\/strong> with the donor red blood cells and look for agglutination.[\/hidden-answer]\r\n\r\nCoombs\u2019 reagent is an antiserum with antibodies that bind to human ________.\r\n[reveal-answer q=\"999685\"]Show Answer[\/reveal-answer]\r\n[hidden-answer a=\"999685\"]Coombs\u2019 reagent is an antiserum with antibodies that bind to human <strong>immunoglobulins, antibodies, and\/or complement<\/strong>.[\/hidden-answer]\r\n\r\n<\/div>\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Think about It<\/h3>\r\n<ol>\r\n \t<li>Explain why the titer of a direct hemagglutination assay is the highest dilution that still causes hemagglutination, whereas in the viral hemagglutination inhibition assay, the titer is the highest dilution at which hemagglutination is not observed.<\/li>\r\n \t<li>Why would a doctor order a direct Coombs\u2019 test when a baby is born with jaundice?<\/li>\r\n<\/ol>\r\n<\/div>\r\n<div class=\"textbox key-takeaways\">\r\n<h3>Critical Thinking<\/h3>\r\nWhen shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type. Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different blood type in critical need of a transfusion? Explain your answers.\r\n\r\n<\/div>","rendered":"<div class=\"textbox learning-objectives\">\n<h3>Learning Objectives<\/h3>\n<ul>\n<li>Compare direct and indirect agglutination<\/li>\n<li>Identify various uses of hemagglutination in the diagnosis of disease<\/li>\n<li>Explain how blood types are determined<\/li>\n<li>Explain the steps used to cross-match blood to be used in a transfusion<\/li>\n<\/ul>\n<\/div>\n<p>In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called <strong>agglutination<\/strong> (<a href=\".\/chapter\/overview-of-specific-adaptive-immunity\/\" target=\"_blank\">Figure\u00a07 in\u00a0Overview of Specific Adaptive Immunity<\/a>). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or <strong>microtiter plate<\/strong> (Figure\u00a01). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.<\/p>\n<div style=\"width: 661px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" class=\"\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172929\/OSC_Microbio_20_03_Microt.jpg\" alt=\"A plastic plate about the size of a hand with wells in a grid. The top is labeled 1-12 and the side is labeled A-H.\" width=\"651\" height=\"488\" data-media-type=\"image\/jpeg\" \/><\/p>\n<p class=\"wp-caption-text\">Figure\u00a01. Microtiter plates are used for conducting numerous reactions simultaneously in an array of wells. (credit: modification of work by &#8220;Microrao&#8221;\/Wikimedia)<\/p>\n<\/div>\n<h2>Agglutination of Bacteria and Viruses<\/h2>\n<p>The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca <strong>Lancefield<\/strong> working with her colleagues A.R. <strong>Dochez<\/strong> and Oswald <strong>Avery<\/strong>.<a class=\"footnote\" title=\"Lancefield, Rebecca C., &quot;The Antigenic Complex of Streptococcus haemoliticus. I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus,&quot; The Journal of Experimental Medicine 47, no. 1 (1928): 91-103.\" id=\"return-footnote-860-1\" href=\"#footnote-860-1\" aria-label=\"Footnote 1\"><sup class=\"footnote\">[1]<\/sup><\/a> She used antibodies to identify <strong>M protein<\/strong>, a virulence factor on streptococci that is necessary for the bacteria\u2019s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.<\/p>\n<p>Lancefield used antisera to show that different strains of the same species of <strong>streptococci<\/strong> express different versions of M protein, which explains why children can come down with <strong>strep throat<\/strong> repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called <strong>serovars<\/strong> because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.<\/p>\n<div id=\"attachment_1735\" style=\"width: 460px\" class=\"wp-caption alignright\"><img loading=\"lazy\" decoding=\"async\" aria-describedby=\"caption-attachment-1735\" class=\"wp-image-1735\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001546\/OSC_Microbio_20_03_Lance1.jpg\" alt=\"Photo of 6 wells. Well 4 has blue spots. All the other wells have a clear blue color.\" width=\"450\" height=\"289\" \/><\/p>\n<p id=\"caption-attachment-1735\" class=\"wp-caption-text\">Figure\u00a02. Antibodies against six different serovars of Group A strep were attached to latex beads. Each of the six antibody preparations was mixed with bacteria isolated from a patient. The tiny clumps seen in well 4 are indicative of agglutination, which is absent from all other wells. This indicates that the serovar associated with well 4 is present in the patient sample. (credit: modification of work by American Society for Microbiology)<\/p>\n<\/div>\n<p>The method developed by Lancefield is a <strong>direct agglutination assay<\/strong>, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert <strong>latex beads<\/strong>. This technique is called an <strong>indirect agglutination assay<\/strong> (or <strong>latex fixation assay<\/strong>), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure\u00a02). Indirect assays can be used to detect the presence of either antibodies or specific antigens.<\/p>\n<p>To identify antibodies in a patient\u2019s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure\u00a03). This technique is most often used when looking for <strong>IgM<\/strong> antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for <strong>rheumatoid factor (RF)<\/strong> to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient\u2019s own <strong>IgG<\/strong>. RF will agglutinate IgG-coated latex beads.<\/p>\n<p>In the reverse test, soluble antigens can be detected in a patient\u2019s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure\u00a03).<\/p>\n<div style=\"width: 1210px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172935\/OSC_Microbio_20_03_Latex.jpg\" alt=\"(a) A diagram of positive agglutination test for antibodies. Large circles (latex beads) have smaller circles (antigens) on their surface. IgM antibodies (6 Y shapes attached at their base) are bound to the antigens. B) A diagram of positive agglutination test for antigens. The latex beads have antigens on them. Smaller blue ovals (pathogens) have circles (antigens) on their surface. The antigens bind to the antibodies.\" width=\"1200\" height=\"670\" data-media-type=\"image\/jpeg\" \/><\/p>\n<p class=\"wp-caption-text\">Figure\u00a03. (a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. (b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies.<\/p>\n<\/div>\n<p>Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the <strong>Widal test<\/strong>, used for the diagnosis of <strong>typhoid fever<\/strong>, looks for agglutination of <strong><em>Salmonella enterica<\/em> subspecies <em>typhi<\/em><\/strong> in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of <em>Salmonella<\/em>, as well as false negatives in patients with hyperproteinemia or immune deficiencies.<\/p>\n<p>In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone <strong>seroconversion<\/strong> when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.<\/p>\n<p>Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient\u2019s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the <strong>titer<\/strong> during the <strong>acute phase<\/strong> of an infection versus the titer from the <strong>convalescent phase<\/strong> will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient\u2019s immune system is responding to the pathogen.<\/p>\n<div class=\"textbox\">\n<p>Watch this video that demonstrates agglutination reactions with latex beads.<\/p>\n<p><iframe loading=\"lazy\" id=\"oembed-1\" title=\"Agglutination assay to detect antigens - Multi-Lingual Captions\" width=\"500\" height=\"375\" src=\"https:\/\/www.youtube.com\/embed\/oXL1rH11MTg?feature=oembed&#38;rel=0\" frameborder=\"0\" allowfullscreen=\"allowfullscreen\"><\/iframe><\/p>\n<\/div>\n<div class=\"textbox key-takeaways\">\n<h3>Think about It<\/h3>\n<ul>\n<li>How is agglutination used to distinguish serovars from each other?<\/li>\n<li>In a latex bead assay to test for antibodies in a patient&#8217;s serum, with what are the beads coated?<\/li>\n<li>What has happened when a patient has undergone seroconversion?<\/li>\n<\/ul>\n<\/div>\n<h3>Hemagglutination<\/h3>\n<p>Agglutination of red blood cells is called <strong>hemagglutination<\/strong>. One common assay that uses hemagglutination is the <strong>direct Coombs\u2019 test<\/strong>, also called the <strong>direct antihuman globulin test (DAT)<\/strong>, which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.<\/p>\n<p>The Coombs\u2019 test is often employed when a newborn has <strong>jaundice<\/strong>, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs\u2019 test is used to determine whether the child\u2019s red blood cells have been bound by the mother\u2019s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs\u2019 tests include <strong>hemolytic transfusion reactions<\/strong>, <strong>autoimmune hemolytic anemia<\/strong>, infectious mononucleosis (caused by <strong>Epstein-Barr virus<\/strong>), <strong>syphilis<\/strong>, and <strong><em>Mycoplasma<\/em> pneumonia<\/strong>. A positive direct Coombs\u2019 test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).<\/p>\n<p>The antibodies bound to red blood cells in these conditions are most often <strong>IgG<\/strong>, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding <strong>Coombs\u2019 reagent<\/strong>, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure\u00a04). The Coombs\u2019 reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.<\/p>\n<p>There is also an <strong>indirect Coombs\u2019 test<\/strong> known as the <strong>indirect antiglobulin test (IAT)<\/strong>. This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum (Figure\u00a04). IAT can be used to screen pregnant women for antibodies that may cause <strong>hemolytic disease of the newborn<\/strong>. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.<\/p>\n<div id=\"attachment_1736\" style=\"width: 610px\" class=\"wp-caption aligncenter\"><a href=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001803\/OSC_Microbio_20_03_Coombs1.jpg\" target=\"_blank\"><img loading=\"lazy\" decoding=\"async\" aria-describedby=\"caption-attachment-1736\" class=\"wp-image-1736\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11001803\/OSC_Microbio_20_03_Coombs1-868x1024.jpg\" alt=\"A) Diagram of direct Coomb\u2019s test. 1. Blood sample from a patient with hemolytic anemia with antibodies attached to red blood cells. 2. Antibodies coat the patient\u2019s red blood cells. 3. A few drops of Coomb\u2019s reagent (containing anti-human antibodies) is mixed in with the patient\u2019s blood sample. 4. Agglutination reaction (clumping) is visible after cross-linkage of antibodies. B) Diagram of indirect Coomb\u2019s test. 1. Patient's serum containing antibodies is drawn. 2. Donor blood is added. 3. Patient's antibodies bind to donor's red blood cells. 4. A few drops of Coombs\u2019 reagent are mixed with the sample. 5. Agglutination reaction is observed.\" width=\"600\" height=\"708\" \/><\/a><\/p>\n<p id=\"caption-attachment-1736\" class=\"wp-caption-text\">Figure\u00a04. Click for a larger image. The steps in direct and indirect Coombs\u2019 tests are shown in the illustration.<\/p>\n<\/div>\n<p>Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, <strong>influenza viruses<\/strong> have two different types of viral spikes called <strong>neuraminidase<\/strong> (N) and <strong>hemagglutinin<\/strong> (H), the latter named for its ability to agglutinate red blood cells (see <a href=\".\/chapter\/viruses\/\" target=\"_blank\">Viruses<\/a>). Thus, we can use red blood cells to detect the presence of influenza virus by <strong>direct hemagglutination assays<\/strong> (HA), in which the virus causes visible agglutination of red blood cells. The <strong>mumps<\/strong> and <strong>rubella<\/strong> viruses can also be detected using HA.<\/p>\n<p>Most frequently, a <strong>serial dilution<\/strong> viral agglutination assay is used to measure the <strong>titer<\/strong> or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure\u00a05).<\/p>\n<div style=\"width: 510px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" class=\"\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172945\/OSC_Microbio_20_03_HA.jpg\" alt=\"A photo of nonagglutination shows a red stripe. A diagram shows that without virus the cells from a compact pellet at the bottom of the well. A photo of agglutination shows a red spot and pink surrounding area. A diagram shows that the red blood cells and viruses form a clump.\" width=\"500\" height=\"447\" data-media-type=\"image\/jpeg\" \/><\/p>\n<p class=\"wp-caption-text\">Figure\u00a05. A viral suspension is mixed with a standardized amount of red blood cells. No agglutination of red blood cells is visible when the virus is absent, and the cells form a compact pellet at the bottom of the well. In the presence of virus, a diffuse pink precipitate forms in the well. (credit bottom: modification of work by American Society for Microbiology)<\/p>\n<\/div>\n<p>A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient\u2019s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a <strong>viral hemagglutination inhibition assay<\/strong> (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient\u2019s serum is the highest dilution that blocks agglutination (Figure\u00a06).<\/p>\n<div id=\"attachment_1737\" style=\"width: 510px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" aria-describedby=\"caption-attachment-1737\" class=\"wp-image-1737\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/11002047\/OSC_Microbio_20_03_HIA1.jpg\" alt=\"A drawing of a well plate. The label at the top reads: Dilution. The rows are labeled: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. The columns are labeled: Sample A, titer = 128 (this spans columns 1 and 2). Sample B, no neutralizing antibody (spans columns 3 and 4). Sample C, titer = 64 spans columns 5 and 6.). Columns 1 and 2 have red dots in all rows but the bottom one. Columns 3 and 4 have no red dots. Columns 5 and 6 have red dots in all rows but the bottom two.\" width=\"500\" height=\"629\" \/><\/p>\n<p id=\"caption-attachment-1737\" class=\"wp-caption-text\">Figure\u00a06. In this HIA, serum containing antibodies to influenzavirus underwent serial two-fold dilutions in a microtiter plate. Red blood cells were then added to the wells. Agglutination only occurred in those wells where the antibodies were too dilute to neutralize the virus. The highest concentration at which agglutination occurs is the titer of the antibodies in the patient\u2019s serum. In the case of this test, Sample A shows a titer of 128, and Sample C shows a titer of 64. (credit: modification of work by Evan Burkala)<\/p>\n<\/div>\n<div class=\"textbox key-takeaways\">\n<h3>Think about It<\/h3>\n<ul>\n<li>What is the mechanism by which viruses are detected in a hemagglutination assay?<\/li>\n<li>Which hemagglutination result tells us the titer of virus in a sample?<\/li>\n<\/ul>\n<\/div>\n<div class=\"textbox shaded\">\n<h3>Animals in the Laboratory<\/h3>\n<p>Much of what we know today about the human immune system has been learned through research conducted using animals\u2014primarily, mammals\u2014as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.<\/p>\n<p>Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.<\/p>\n<p>We also use animals for the study of disease. The only way to grow <em>Treponema pallidum<\/em> for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch\u2019s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.<\/p>\n<p>While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one\u2014indeed, one of Charles Darwin&#8217;s daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document &#8220;Guide for the Care and Use of Laboratory Animals&#8221; makes it clear that the use of animals in research is a privilege granted by society to researchers.<\/p>\n<p>The NIH guidelines are based on the principle of the three R\u2019s: replace, refine, and reduce. Researchers should strive to <em>replace<\/em> animal models with nonliving models, <em>replace<\/em> vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should <em>refine<\/em> husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that <em>reduce<\/em> the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.<\/p>\n<p>At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.<\/p>\n<\/div>\n<div class=\"textbox\">Visit this site to <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK54050\/\" target=\"_blank\">view the NIH Guide for the Care and Use of Laboratory Animals<\/a>.<\/div>\n<h2>Blood Typing and Cross-Matching<\/h2>\n<p>In addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against <strong>blood types<\/strong> other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the <strong>ABO<\/strong> and <strong>Rh blood groups<\/strong> (see <a href=\".\/chapter\/hypersensitivities\/\" target=\"_blank\">Figure\u00a03 in\u00a0Hypersensitivities<\/a>).<\/p>\n<p>When units of blood are being considered for <strong>transfusion<\/strong>, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient\u2019s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure\u00a07, if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative.<\/p>\n<p>Following determination of the blood type, immediately prior to releasing the blood for transfusion, a <strong>cross-match<\/strong> is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, <strong>hemagglutination<\/strong> will occur. To confirm any negative test results and check for sensitized red blood cells, <strong>Coombs\u2019 reagent<\/strong> may be added to the mix to facilitate visualization of the antibody-red blood cell interaction.<\/p>\n<p>Under some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation.<\/p>\n<p>Red blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, women who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an <strong>antibody screen<\/strong> test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit.<\/p>\n<div style=\"width: 1107px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/s3-us-west-2.amazonaws.com\/courses-images\/wp-content\/uploads\/sites\/1094\/2016\/11\/03172951\/OSC_Microbio_20_03_BloodT.jpg\" alt=\"Three wells the first well (labeled anti-A) shows black spots and is labeled clumping. This is due to the agglutinated RBCs. The second well (labeled anti-B) looks smooth and has no clumping. The third well (labeled) anti-Rh shows clumping because it has spots.\" width=\"1097\" height=\"350\" data-media-type=\"image\/jpeg\" \/><\/p>\n<p class=\"wp-caption-text\">Figure\u00a07. This sample of a commercially produced &#8220;bedside&#8221; card enables quick typing of both a recipient\u2019s and donor\u2019s blood before transfusion. The card contains three reaction sites or wells. One is coated with an anti-A antibody, one with an anti-B antibody, and one with an anti-Rh antibody. Agglutination of red blood cells in a given site indicates a positive identification of the blood antigens: in this case, A and Rh antigens for blood type A-positive.<\/p>\n<\/div>\n<div class=\"textbox key-takeaways\">\n<h3>Think about It<\/h3>\n<ul>\n<li>If a patient&#8217;s blood agglutinates with anti-B serum, what is the patient\u2019s blood type?<\/li>\n<li>What is a cross-match assay, and why is it performed?<\/li>\n<\/ul>\n<\/div>\n<p>Table 1 summarizes the various kinds of agglutination assays discussed in this section.<\/p>\n<table id=\"fs-id1167661289772\" class=\"span-all\" summary=\"Table titled: Mechanisms of Select Antibody-Antigen Assays. Type of Assay: Agglutination. Mechanism: Direct: Antibody is used to clump bacterial cells or other large structures. Example: Serotyping bacteria. Mechanism: Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum. Example: Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum. Type of assay: Hemagglutination. Mechanism: Direct: Some bacteria and viruses cross-link red blood cells and clump them together. Example: Diagnosing influenza, mumps, and measles. Mechanism: Direct Coombs\u2019 test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells in vivo. Example: Checking for maternal antibodies binding to neonatal red blood cells. Mechanism: Indirect Coombs\u2019 test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum in vitro. Example: Performing pretransfusion blood testing. Mechanism: Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination. Example: Diagnosing various viral diseases by the presence of patient antibodies against the virus. Mechanism: Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood. Example: Matches donor blood to recipient immune requirements.\">\n<thead>\n<tr>\n<th colspan=\"3\" data-align=\"center\">Table 1. Mechanisms of Select Antibody-Antigen Assays<\/th>\n<\/tr>\n<tr valign=\"top\">\n<th data-valign=\"top\" data-align=\"left\">Type of Assay<\/th>\n<th data-valign=\"top\" data-align=\"left\">Mechanism<\/th>\n<th data-valign=\"top\" data-align=\"left\">Example<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr valign=\"top\">\n<td rowspan=\"2\" data-valign=\"top\" data-align=\"left\">Agglutination<\/td>\n<td data-valign=\"top\" data-align=\"left\">Direct: Antibody is used to clump bacterial cells or other large structures<\/td>\n<td data-valign=\"top\" data-align=\"left\">Serotyping bacteria<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td data-valign=\"top\" data-align=\"left\">Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum<\/td>\n<td data-valign=\"top\" data-align=\"left\">Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td rowspan=\"5\" data-valign=\"top\" data-align=\"left\">Hemagglutination<\/td>\n<td data-valign=\"top\" data-align=\"left\">Direct: Some bacteria and viruses cross-link red blood cells and clump them together<\/td>\n<td data-valign=\"top\" data-align=\"left\">Diagnosing influenza, mumps, and measles<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td data-valign=\"top\" data-align=\"left\">Direct Coombs\u2019 test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells <em>in vivo<\/em><\/td>\n<td data-valign=\"top\" data-align=\"left\">Checking for maternal antibodies binding to neonatal red blood cells<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td data-valign=\"top\" data-align=\"left\">Indirect Coombs\u2019 test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient\u2019s serum <em>in vitro<\/em><\/td>\n<td data-valign=\"top\" data-align=\"left\">Performing pretransfusion blood testing<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td data-valign=\"top\" data-align=\"left\">Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination<\/td>\n<td data-valign=\"top\" data-align=\"left\">Diagnosing various viral diseases by the presence of patient antibodies against the virus<\/td>\n<\/tr>\n<tr valign=\"top\">\n<td data-valign=\"top\" data-align=\"left\">Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood<\/td>\n<td data-valign=\"top\" data-align=\"left\">Matches donor blood to recipient immune requirements<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<div class=\"textbox key-takeaways\">\n<h3>Key Concepts and Summary<\/h3>\n<ul>\n<li>Antibodies can agglutinate cells or large particles into a visible matrix. <strong>Agglutination<\/strong> tests are often done on cards or in <strong>microtiter plates<\/strong> that allow multiple reactions to take place side by side using small volumes of reagents.<\/li>\n<li>Using antisera against certain proteins allows identification of <strong>serovars<\/strong> within species of bacteria.<\/li>\n<li>Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through <strong>seroconversion<\/strong> and the level of antibodies becomes detectable.<\/li>\n<li>Agglutination of latex beads in <strong>indirect agglutination assays<\/strong> can be used to detect the presence of specific antigens or specific antibodies in patient serum.<\/li>\n<li>The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct <strong>Coombs\u2019 test<\/strong>, which uses Coombs\u2019 reagent to cross-link antibodies bound to red blood cells and facilitate <strong>hemagglutination<\/strong>.<\/li>\n<li>Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the <strong>direct hemagglutination assay<\/strong>, most often used to determine the titer of virus in solution.<\/li>\n<li><strong>Neutralization assays<\/strong> quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.<\/li>\n<li>Hemagglutination assays are also used to screen and <strong>cross-match<\/strong> donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.<\/li>\n<\/ul>\n<\/div>\n<div class=\"textbox exercises\">\n<h3>Multiple Choice<\/h3>\n<p>We use antisera to distinguish between various ________ within a species of bacteria.<\/p>\n<ol style=\"list-style-type: lower-alpha;\">\n<li>isotypes<\/li>\n<li>serovars<\/li>\n<li>subspecies<\/li>\n<li>lines<\/li>\n<\/ol>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q443301\">Show Answer<\/span><\/p>\n<div id=\"q443301\" class=\"hidden-answer\" style=\"display: none\">Answer b.\u00a0We use antisera to distinguish between various serovars within a species of bacteria.<\/div>\n<\/div>\n<p>When using antisera to characterize bacteria, we will often link the antibodies to ________ to better visualize the agglutination.<\/p>\n<ol style=\"list-style-type: lower-alpha;\">\n<li>latex beads<\/li>\n<li>red blood cells<\/li>\n<li>other bacteria<\/li>\n<li>white blood cells<\/li>\n<\/ol>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q71926\">Show Answer<\/span><\/p>\n<div id=\"q71926\" class=\"hidden-answer\" style=\"display: none\">Answer a.\u00a0When using antisera to characterize bacteria, we will often link the antibodies to latex beads to better visualize the agglutination.<\/div>\n<\/div>\n<p>The antibody screening test that is done along with pretransfusion blood typing is used to ensure that the recipient<\/p>\n<ol style=\"list-style-type: lower-alpha;\">\n<li>does not have a previously undetected bacterial or viral infection.<\/li>\n<li>is not immunocompromised.<\/li>\n<li>actually does have the blood type stated in the online chart.<\/li>\n<li>is not making antibodies against antigens outside the ABO or Rh systems.<\/li>\n<\/ol>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q92382\">Show Answer<\/span><\/p>\n<div id=\"q92382\" class=\"hidden-answer\" style=\"display: none\">Answer d. This test is done to ensure\u00a0that the recipient is not making antibodies against antigens outside the ABO or Rh systems.<\/div>\n<\/div>\n<p>The direct Coombs\u2019 test is designed to detect when people have a disease that causes them to<\/p>\n<ol style=\"list-style-type: lower-alpha;\">\n<li>have an excessively high fever.<\/li>\n<li>quit making antibodies.<\/li>\n<li>make too many red blood cells.<\/li>\n<li>produce antibodies that bind to their own red blood cells.<\/li>\n<\/ol>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q511979\">Show Answer<\/span><\/p>\n<div id=\"q511979\" class=\"hidden-answer\" style=\"display: none\">Answer d. The direct Coombs\u2019 test is designed to detect when people have a disease that causes them to\u00a0produce antibodies that bind to their own red blood cells.<\/div>\n<\/div>\n<p>Viral hemagglutination assays only work with certain types of viruses because<\/p>\n<ol style=\"list-style-type: lower-alpha;\">\n<li>the virus must be able to cross-link red blood cells directly.<\/li>\n<li>the virus must be able to lyse red blood cells.<\/li>\n<li>the virus must not be able to lyse red blood cells.<\/li>\n<li>other viruses are too dangerous to work with in a clinical lab setting.<\/li>\n<\/ol>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q862143\">Show Answer<\/span><\/p>\n<div id=\"q862143\" class=\"hidden-answer\" style=\"display: none\">Answer a.\u00a0Viral hemagglutination assays only work with certain types of viruses because\u00a0the virus must be able to cross-link red blood cells directly.<\/div>\n<\/div>\n<\/div>\n<div class=\"textbox exercises\">\n<h3>Fill in the Blank<\/h3>\n<p>In the major cross-match, we mix ________ with the donor red blood cells and look for agglutination.<\/p>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q903843\">Show Answer<\/span><\/p>\n<div id=\"q903843\" class=\"hidden-answer\" style=\"display: none\">In the major cross-match, we mix <strong>patient serum<\/strong> with the donor red blood cells and look for agglutination.<\/div>\n<\/div>\n<p>Coombs\u2019 reagent is an antiserum with antibodies that bind to human ________.<\/p>\n<div class=\"qa-wrapper\" style=\"display: block\"><span class=\"show-answer collapsed\" style=\"cursor: pointer\" data-target=\"q999685\">Show Answer<\/span><\/p>\n<div id=\"q999685\" class=\"hidden-answer\" style=\"display: none\">Coombs\u2019 reagent is an antiserum with antibodies that bind to human <strong>immunoglobulins, antibodies, and\/or complement<\/strong>.<\/div>\n<\/div>\n<\/div>\n<div class=\"textbox key-takeaways\">\n<h3>Think about It<\/h3>\n<ol>\n<li>Explain why the titer of a direct hemagglutination assay is the highest dilution that still causes hemagglutination, whereas in the viral hemagglutination inhibition assay, the titer is the highest dilution at which hemagglutination is not observed.<\/li>\n<li>Why would a doctor order a direct Coombs\u2019 test when a baby is born with jaundice?<\/li>\n<\/ol>\n<\/div>\n<div class=\"textbox key-takeaways\">\n<h3>Critical Thinking<\/h3>\n<p>When shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type. Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different blood type in critical need of a transfusion? Explain your answers.<\/p>\n<\/div>\n\n\t\t\t <section class=\"citations-section\" role=\"contentinfo\">\n\t\t\t <h3>Candela Citations<\/h3>\n\t\t\t\t\t <div>\n\t\t\t\t\t\t <div id=\"citation-list-860\">\n\t\t\t\t\t\t\t <div class=\"licensing\"><div class=\"license-attribution-dropdown-subheading\">CC licensed content, Shared previously<\/div><ul class=\"citation-list\"><li>OpenStax Microbiology. <strong>Provided by<\/strong>: OpenStax CNX. <strong>Located at<\/strong>: <a target=\"_blank\" href=\"http:\/\/cnx.org\/contents\/e42bd376-624b-4c0f-972f-e0c57998e765@4.2\">http:\/\/cnx.org\/contents\/e42bd376-624b-4c0f-972f-e0c57998e765@4.2<\/a>. <strong>License<\/strong>: <em><a target=\"_blank\" rel=\"license\" href=\"https:\/\/creativecommons.org\/licenses\/by\/4.0\/\">CC BY: Attribution<\/a><\/em>. <strong>License Terms<\/strong>: Download for free at http:\/\/cnx.org\/contents\/e42bd376-624b-4c0f-972f-e0c57998e765@4.2<\/li><\/ul><\/div>\n\t\t\t\t\t\t <\/div>\n\t\t\t\t\t <\/div>\n\t\t\t <\/section><hr class=\"before-footnotes clear\" \/><div class=\"footnotes\"><ol><li id=\"footnote-860-1\">Lancefield, Rebecca C., \"The Antigenic Complex of <em>Streptococcus haemoliticus<\/em>. I. Demonstration of a Type-Specific Substance in Extracts of <em>Streptococcus haemolyticus<\/em>,\" <em>The Journal of Experimental Medicine<\/em> 47, no. 1 (1928): 91-103. <a href=\"#return-footnote-860-1\" class=\"return-footnote\" aria-label=\"Return to footnote 1\">&crarr;<\/a><\/li><\/ol><\/div>","protected":false},"author":17,"menu_order":4,"template":"","meta":{"_candela_citation":"[{\"type\":\"cc\",\"description\":\"OpenStax Microbiology\",\"author\":\"\",\"organization\":\"OpenStax CNX\",\"url\":\"http:\/\/cnx.org\/contents\/e42bd376-624b-4c0f-972f-e0c57998e765@4.2\",\"project\":\"\",\"license\":\"cc-by\",\"license_terms\":\"Download for free at http:\/\/cnx.org\/contents\/e42bd376-624b-4c0f-972f-e0c57998e765@4.2\"}]","CANDELA_OUTCOMES_GUID":"","pb_show_title":"on","pb_short_title":"","pb_subtitle":"","pb_authors":[],"pb_section_license":""},"chapter-type":[],"contributor":[],"license":[],"class_list":["post-860","chapter","type-chapter","status-publish","hentry"],"part":833,"_links":{"self":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapters\/860","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapters"}],"about":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/wp\/v2\/types\/chapter"}],"author":[{"embeddable":true,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/wp\/v2\/users\/17"}],"version-history":[{"count":3,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapters\/860\/revisions"}],"predecessor-version":[{"id":1738,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapters\/860\/revisions\/1738"}],"part":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/parts\/833"}],"metadata":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapters\/860\/metadata\/"}],"wp:attachment":[{"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/wp\/v2\/media?parent=860"}],"wp:term":[{"taxonomy":"chapter-type","embeddable":true,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/pressbooks\/v2\/chapter-type?post=860"},{"taxonomy":"contributor","embeddable":true,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/wp\/v2\/contributor?post=860"},{"taxonomy":"license","embeddable":true,"href":"https:\/\/courses.lumenlearning.com\/suny-microbiology\/wp-json\/wp\/v2\/license?post=860"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}