Antibody elution

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An antibody elution removes bound antibody from the surface of a red blood cell to aid in the antibody identification process. Elution principle.jpg
An antibody elution removes bound antibody from the surface of a red blood cell to aid in the antibody identification process.

An antibody elution is a clinical laboratory diagnostic procedure which removes sensitized antibodies from red blood cells, in order to determine the blood group system antigen the antibody targets. [1] An antibody elution is deemed necessary when antibodies of the immunoglobulin class G (IgG) are found sensitized (bound) to peripheral red cells collected from a blood product transfusion recipient. [2] IgG antibodies are detected using an assay known as the direct antiglobulin test. [3]

Contents

Antibody elutions are specialized tests used in clinical blood banks. Examples of routine tests include ABO/Rh, antibody screen, antibody identification, and antiglobulin testing. Examples of other specialized tests used in blood banking include: treatment with thiol reagent, monocyte monolayer assay, enzyme treatment, and adsorptions. [2]

This procedure aids in the investigation of antibodies that are difficult to identify, distinguishing transfusion reactions, hemolytic disease of the fetus and newborn, and warm autoantibody workups. [4]

Background

Blood group systems

Red blood cell membranes consist of a phospholipid bilayer, littered with proteins, lipids, carbohydrates, and combinations of these substances. [5] These substances are called antigens because they stimulate an immune response when an individual is exposed to the substance, but the exposed individual does not carry nor express the genes which encode said antigens. [6] Each individual has a unique genetic and phenotypic makeup of antigens, much like the dermatoglyphics of human fingerprints.

As of 2023, there are 44 blood group systems, each containing several red blood cell antigens totaling 354, determined by approximately 49 separate genes. [7] Of these antigens, only a handful are considered clinically significant, meaning that they can stimulate the production of antibodies capable of causing red cell hemolysis. This is particularly important for the transfusion of packed red blood cells and other cellular blood products. Examples of blood group systems that contain antigens capable of inducing clinically significant alloantibodies (antibodies against non-self antigens) include, but are not limited to the ABO, Rh, Kell, Duffy, Kidd, and MNS blood group systems. [8]

Antibody identification

Antibodies to blood group system antigens and their characteristics must be identified when such antibodies are detected in a potential recipient's serum or plasma. [9] The specificity of the antibody aids the medical laboratory scientist in determining if the antibody is clinically significant. Antibody identification is a very laborious process. [1]

Characteristics of clinically significant antibodies include: reactive at body temperature (37°C), immunoglobulin (Ig) class G, IgM that reacts at body temperature, ability to cross the placenta, ability to cause red blood cell destruction, and/or antibodies directed against commonly known clinically significant red cell antigens. [10] For example, if an individual is exposed to a red cell antigen (via blood transfusion, pregnancy, stem-cell transplant) that they do not inherently possess, they may form a clinically significant antibody directed against that antigen. If a patient receives a transfusion of packed red blood cells possessing the Kell antigen (big K or simply K), they may form an antibody called anti-K (anti big K). Subsequent transfusions with K-positive packed red blood cells would cause an immediate hemolytic transfusion reaction. The K antibody reacts at 37°C, is IgG, capable of crossing the placenta, and known to cause immediate red blood cell destruction.

The presence of autoantibodies directed against self red blood cell antigens can complicate the antibody identification process. Red blood cell autoantibodies tend to be specific for red cell antigens of high frequency within the population. An antibody elution can aid in the identification of clinically significant alloantibodies when autoantibodies interfere with the antibody identification process. [11]

Methods of antibody elution

There are several methods of antibody elution used in clinical blood banking. Some of these methods include manipulating temperature, manipulating pH, use of organic solvents, and chloroquine. [2] Each of these methods have advantages and disadvantages, and the method of elution will vary depending on clinical utility. One of the more commonly used methods is an acid elution, because it is quick, cheap, and relatively easy to perform. [3] [12]

Acid elution principle

The main steps involved in an acid elution include: [3] [2]

  1. Separate and thoroughly wash red blood cells from a peripherally collected EDTA blood collection tube using centrifugation.
  2. Mix washed patient red blood cells, that are positive for the IgG phase of the direct antiglobulin test, with glycine acid (pH 3.0).
  3. Centrifuge the mixture, and immediately remove the supernatant from the destroyed red blood cells.
  4. Add buffer to return the mixture to a neutral pH. This step is critical for further antibody identification testing, because the antibody will not react at a pH of less than 7.0.
  5. Additional centrifugation may be needed to clarify the solution.
  6. The resulting solution is known as the eluate. This eluate is then tested against a panel of red blood cells with known antigen profiles. This antibody identification procedure will aid in determining the specificity of the antibody.
This infographic shows the acid antibody elution principle. The Acid Elution Principle.jpg
This infographic shows the acid antibody elution principle.

Determining when to perform an antibody elution

Antiglobulin testing

The main method of antibody and antigen detection used in a clinical laboratory is red blood cell agglutination. [1] Most IgM antibodies are easier to detect because they are larger and react at room temperature (20°C). [13] [14] This concept is what makes ABO/Rh testing so quick and easy to perform. However, most clinically significant non-ABO antibodies react at body temperature (37°C) and will not result in agglutination without the addition of multiple steps: incubation, washing, and the addition of anti-human globulin (AHG) reagent. [15]

Anti-human globulin is an antibody directed against human IgG antibodies. [16] When the smaller IgG antibody is attached to red blood cells, the larger AHG antibodies create a cross-link between IgG sensitized RBC forming visual agglutination. When this agglutination is observed, the antiglobulin test is considered positive for the detection of the antibody and/or antigen(s) present. [2]

There are two main types of antiglobulin testing: indirect and direct. [17] [18] Indirect antiglobulin testing is used to detect antibodies in plasma/serum, whereas direct antiglobulin testing is used to detect antibody bound to red blood cells. When the direct antiglobulin test is positive, we must perform an antibody elution to remove the antibody for identification and to determine the antibody's clinical significance. [3]

See also

Related Research Articles

<span class="mw-page-title-main">Blood type</span> Classification of blood based on antibodies and antigens on red blood cell surfaces

A blood type is a classification of blood, based on the presence and absence of antibodies and inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system. Some of these antigens are also present on the surface of other types of cells of various tissues. Several of these red blood cell surface antigens can stem from one allele and collectively form a blood group system.

Serology is the scientific study of serum and other body fluids. In practice, the term usually refers to the diagnostic identification of antibodies in the serum. Such antibodies are typically formed in response to an infection, against other foreign proteins, or to one's own proteins. In either case, the procedure is simple.

<span class="mw-page-title-main">Hemolytic disease of the newborn</span> Fetal and neonatal alloimmune blood condition

Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis foetalis, is an alloimmune condition that develops in a fetus at or around birth, when the IgG molecules produced by the mother pass through the placenta. Among these antibodies are some which attack antigens on the red blood cells in the fetal circulation, breaking down and destroying the cells. The fetus can develop reticulocytosis and anemia. The intensity of this fetal disease ranges from mild to very severe, and fetal death from heart failure can occur. When the disease is moderate or severe, many erythroblasts are present in the fetal blood, earning these forms of the disease the name erythroblastosis fetalis.

A Coombs test, also known as antiglobulin test (AGT), is either of two blood tests used in immunohematology. They are the direct and indirect Coombs tests. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. Since these antibodies sometimes destroy red blood cells, a person can be anemic and this test can help clarify the condition. The indirect Coombs detects antibodies that are floating freely in the blood. These antibodies could act against certain red blood cells and the test can be done to diagnose reactions to a blood transfusion.

<span class="mw-page-title-main">Cross-matching</span> Testing before a blood transfusion

Cross-matching or crossmatching is a test performed before a blood transfusion as part of blood compatibility testing. Normally, this involves adding the recipient's blood plasma to a sample of the donor's red blood cells. If the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. This antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with anti-human globulin. Along with blood typing of the donor and recipient and screening for unexpected blood group antibodies, cross-matching is one of a series of steps in pre-transfusion testing. In some circumstances, an electronic cross-match can be performed by comparing records of the recipient's ABO and Rh blood type against that of the donor sample. In emergencies, blood may be issued before cross-matching is complete. Cross-matching is also used to determine compatibility between a donor and recipient in organ transplantation.

Autoimmune hemolytic anemia (AIHA) occurs when antibodies directed against the person's own red blood cells (RBCs) cause them to burst (lyse), leading to an insufficient number of oxygen-carrying red blood cells in the circulation. The lifetime of the RBCs is reduced from the normal 100–120 days to just a few days in serious cases. The intracellular components of the RBCs are released into the circulating blood and into tissues, leading to some of the characteristic symptoms of this condition. The antibodies are usually directed against high-incidence antigens, therefore they also commonly act on allogenic RBCs. AIHA is a relatively rare condition, with an incidence of 5–10 cases per 1 million persons per year in the warm-antibody type and 0.45 to 1.9 cases per 1 million persons per year in the cold antibody type. Autoimmune hemolysis might be a precursor of later onset systemic lupus erythematosus.

Paroxysmal cold hemoglobinuria (PCH) is an autoimmune hemolytic anemia featured by complement-mediated intravascular hemolysis after cold exposure. It can present as an acute non-recurrent postinfectious event in children, or chronic relapsing episodes in adults with hematological malignancies or tertiary syphilis. Described by Julius Donath (1870–1950) and Karl Landsteiner (1868–1943) in 1904, PCH is one of the first clinical entities recognized as an autoimmune disorder.

In ABO hemolytic disease of the newborn maternal IgG antibodies with specificity for the ABO blood group system pass through the placenta to the fetal circulation where they can cause hemolysis of fetal red blood cells which can lead to fetal anemia and HDN. In contrast to Rh disease, about half of the cases of ABO HDN occur in a firstborn baby and ABO HDN does not become more severe after further pregnancies.

Hemolytic disease of the newborn (anti-Rhc) can range from a mild to a severe disease. It is the third most common cause of severe HDN. Rh disease is the most common and hemolytic disease of the newborn (anti-Kell) is the second most common cause of severe HDN. It occurs more commonly in women who are Rh D negative.

The term human blood group systems is defined by the International Society of Blood Transfusion (ISBT) as systems in the human species where cell-surface antigens—in particular, those on blood cells—are "controlled at a single gene locus or by two or more very closely linked homologous genes with little or no observable recombination between them", and include the common ABO and Rh (Rhesus) antigen systems, as well as many others; 44 human systems are identified as of December 2022.

The Kidd antigen system are proteins found in the Kidd's blood group, which act as antigens, i.e., they have the ability to produce antibodies under certain circumstances. The Jk antigen is found on a protein responsible for urea transport in the red blood cells and the kidney. They are important in transfusion medicine. People with two Jk(a) antigens, for instance, may form antibodies against donated blood containing two Jk(b) antigens. This can lead to hemolytic anemia, in which the body destroys the transfused blood, leading to low red blood cell counts. Another disease associated with the Jk antigen is hemolytic disease of the newborn, in which a pregnant woman's body creates antibodies against the blood of her fetus, leading to destruction of the fetal blood cells. Hemolytic disease of the newborn associated with Jk antibodies is typically mild, though fatal cases have been reported.

<span class="mw-page-title-main">Ii antigen system</span> Human blood group system

The Ii antigen system is a human blood group system based upon a gene on chromosome 6 and consisting of the I antigen and the i antigen. The I antigen is normally present on the cell membrane of red blood cells in all adults, while the i antigen is present in fetuses and newborns.

<span class="mw-page-title-main">Neonatal isoerythrolysis</span> Blood disorder in newborn kittens and foals

Neonatal isoerythrolysis (NI), also known as hemolytic icterus or hemolytic anemia, is a disease most commonly seen in kittens and foals, but has also been reported in puppies. It occurs when the mother has antibodies against the blood type of the newborn.

The LW blood system was first described by Landsteiner and Wiener in 1940. It was often confused with the Rh system, not becoming a separate antigen system until 1982. The LW and RhD antigens are genetically independent though they are phenotypically related and the LW antigen is expressed more strongly on RhD positive cells than on RhD negative cells. In most populations, the antithetical LW antigens, LWa and LWb are present as very high and very low frequency, respectively.

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<span class="mw-page-title-main">Red cell agglutination</span> Clumping of red blood cells

In hematology, red cell agglutination or autoagglutination is a phenomenon in which red blood cells clump together, forming aggregates. It is caused by the surface of the red cells being coated with antibodies. This often occurs in cold agglutinin disease, a type of autoimmune hemolytic anemia in which people produce antibodies that bind to their red blood cells at cold temperatures and destroy them. People may develop cold agglutinins from lymphoproliferative disorders, from infection with Mycoplasma pneumoniae or Epstein–Barr virus, or idiopathically. Red cell agglutination can also occur in paroxysmal nocturnal hemoglobinuria and warm autoimmune hemolytic anemia. In cases of red cell agglutination, the direct antiglobulin test can be used to demonstrate the presence of antibodies bound to the red cells.

Thermal amplitude or thermal range refers to the temperature range in which a cold autoantibody or cold-reacting alloantibody binds to its antigen. Cold antibodies that can bind to antigen above 30 °C (86 °F) are considered potentially clinically significant and may lead to disease that occurs or worsens on exposure to low temperatures. The closer the thermal range comes to core body temperature, the greater the chance that the antibody will cause symptoms such as anemia or Raynaud syndrome. Antibodies that are only reactive at temperatures below 30 °C (86 °F) are generally considered unlikely to be clinically significant.

<span class="mw-page-title-main">Blood compatibility testing</span> Testing to identify incompatibilities between blood types

Blood compatibility testing is conducted in a medical laboratory to identify potential incompatibilities between blood group systems in blood transfusion. It is also used to diagnose and prevent some complications of pregnancy that can occur when the baby has a different blood group from the mother. Blood compatibility testing includes blood typing, which detects the antigens on red blood cells that determine a person's blood type; testing for unexpected antibodies against blood group antigens ; and, in the case of blood transfusions, mixing the recipient's plasma with the donor's red blood cells to detect incompatibilities (crossmatching). Routine blood typing involves determining the ABO and RhD type, and involves both identification of ABO antigens on red blood cells and identification of ABO antibodies in the plasma. Other blood group antigens may be tested for in specific clinical situations.

The Lan blood group system is a human blood group defined by the presence or absence of the Lan antigen on a person's red blood cells. More than 99.9% of people are positive for the Lan antigen. Individuals with the rare Lan-negative blood type, which is a recessive trait, can produce an anti-Lan antibody when exposed to Lan-positive blood. Anti-Lan antibodies may cause transfusion reactions on subsequent exposures to Lan-positive blood, and have also been implicated in mild cases of hemolytic disease of the newborn. However, the clinical significance of the antibody is variable. The antigen was first described in 1961, and Lan was officially designated a blood group in 2012.

<span class="mw-page-title-main">Monocyte monolayer assay</span> Laboratory test for clinically significant alloantibodies

The monocyte monolayer assay (MMA) is used to determine the clinical significance of alloantibodies produced by blood transfusion recipients. The assay is used to assess the potential for intravascular hemolysis when incompatible cellular blood products are transfused to the anemic patient. When donor cells possess substances that are not produced by the recipient, the recipient's immune system produces antibodies against the substance; these are called alloantibodies. Specific white blood cells, called monocytes, are tasked with ingesting foreign material and become activated during certain inflammatory events. These activated monocytes come in contact with antibody-sensitized red blood cells (RBC) and may or may not exhibit phagocytosis (ingestion) and destroy the donor red blood cells. If monocytes destroy the RBC, the antibody attached to those RBC is considered clinically significant.

References

  1. 1 2 3 Modern blood banking & transfusion practices. Denise Harmening (Seventh ed.). Philadelphia, PA. 2019. ISBN   978-0-8036-9462-0. OCLC   1048659017.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  2. 1 2 3 4 5 Technical manual. Caludia S. Cohn, Meghan Delaney, Susan T. Johnson, Louis M. Katz, American Association of Blood Banks (20th ed.). Bethesda, Maryland. 2020. ISBN   978-1-56395-370-5. OCLC   1163631267.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  3. 1 2 3 4 Judd, W. John (2021). Judd's methods in immunohematology. Susan T. Johnson, Jill Storry, W. John Judd, American Association of Blood Banks (Fourth ed.). Bethesda, MD. ISBN   978-1-56395-435-1. OCLC   1336509285.{{cite book}}: CS1 maint: location missing publisher (link)
  4. ↑ "Glossary: Elution - Blood Bank Guy Glossary". Blood Bank Guy. Retrieved 2023-04-17.
  5. ↑ Li, He; Lykotrafitis, George (2014-08-05). "Erythrocyte membrane model with explicit description of the lipid bilayer and the spectrin network". Biophysical Journal. 107 (3): 642–653. Bibcode:2014BpJ...107..642L. doi:10.1016/j.bpj.2014.06.031. ISSN   1542-0086. PMC   4129483 . PMID   25099803.
  6. ↑ Miller, Linda E. (2021). Clinical immunology and serology : a laboratory perspective. Christine Dorresteyn Stevens (Fifth ed.). Philadelphia, PA. ISBN   978-0-8036-9440-8. OCLC   1200832451.{{cite book}}: CS1 maint: location missing publisher (link)
  7. ↑ ISBT. "Red Cell Immunogenetics and Blood Group Terminology | ISBT Working Party". www.isbtweb.org. Retrieved 2023-04-10.
  8. ↑ "Clinically Significant Red Cell Antibodies". Bloodworks Northwest. Retrieved 2023-04-10.
  9. ↑ QUINLEY, EVA D. (2020). IMMUNOHEMATOLOGY : principles and practice. [S.l.]: JONES & BARTLETT LEARNING. ISBN   978-1-284-22137-4. OCLC   1162875932.
  10. ↑ White, J. (February 2017). "Red cell antibodies - clinical significance or just noise?". ISBT Science Series. 12 (1): 19–24. doi:10.1111/voxs.12325. S2CID   78254504.
  11. ↑ "Glossary: Autoantibody - Blood Bank Guy Glossary". Blood Bank Guy. Retrieved 2023-04-17.
  12. ↑ "Glossary: Elution - Blood Bank Guy Glossary". Blood Bank Guy. Retrieved 2023-04-17.
  13. ↑ "Glossary: IgM". Blood Bank Guy. Retrieved 2023-04-20.
  14. ↑ "Glossary: IgG". Blood Bank Guy. Retrieved 2023-04-20.
  15. ↑ Blaney, Kathy D. (2013). Basic & applied concepts of blood banking and transfusion practices. Paula R. Howard, Kathy D. Blaney (3rd ed.). St. Louis, Mo.: Mosby. ISBN   978-0-323-08663-9. OCLC   817741300.
  16. ↑ "Glossary: AHG Phase - Blood Bank Guy Glossary". Blood Bank Guy. Retrieved 2023-04-20.
  17. ↑ "Glossary: Direct Antiglobulin Test - Blood Bank Guy Glossary". Blood Bank Guy. Retrieved 2023-04-20.
  18. ↑ "Glossary: Indirect Antiglobulin Test". Blood Bank Guy. Retrieved 2023-04-20.
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