> See also: > - Reference --- Review: - **Agglutination:** The clumping together of small particles suspended in a solution --- # The Coombs Test (AHG Test) - **Alternative Names:** - Antiglobulin Test - Antihuman Globulin Test (AHG) - Indirect/Direct ( ) Test (IAT/DAT) ### Summary The purpose of the **Coombs test** is to *overcome IgG's inability to agglutinate* (clump together) within a solution regardless of if it has/hasn't successfully bound to its target. This test solves this issue through the use of **antihuman globulin (AHG)**: - It is isotype *IgM* and it's *pentameric* structure is what makes it useful - Unlike other isotypes, the unique pentameric structure of IgM allows it to readily agglutinate. - Is also called *Anti-IgG* as it binds to the Fc region of any IgG immunoglobulin, regardless of specificity. There are three relevant antibodies in this test: - An IgG - IgM: Neither the known or unknown, it is a sort of mediator that allows the known IgG antibody to agglutinate ### Associated Diseases > **Anemia:** A condition marked by a deficiency of red blood cells or of hemoglobin in the blood - *Hemolytic Disease of the Newborn (HDL)*: A mother's antibodies (IgG) are passed through the placenta and attach onto the babies red blood cells causing anemia - *Autoimmune Hemolytic Anemia (AIHA)*: a ## Procedure > [!info]- **Knowns vs Unknowns** > **Known:** Antibody > > *Anti-human globulin (AHG)*, or antiglobulin, is the *known antibody* of both the direct and indirect tests. It is of the isotype IgM and attached the the Fc (constant) region of > - *Isotype IgM* > > --- > **Unknown:** Antigen (IgG) > > --- > - Both the knowns and unknowns in this experiment are IgG 1. IgG antibodies are - In the direct test, IgG antibodies are introduced into the baby's 2. Anti-Human Globulin (AHG) is then added which will bind The general procedure is to treat the sample with AHG (Anti-IgG). #### Washing Process > Saline (Normal Saline, 0.9% Saline, Physiological Saline) When washing, you centrifuge for **60 seconds after EACH wash** - A total of *3 times* while washing for *both the direct/indirect* procedures - An overall time of 180 seconds for the whole washing step, followed by an additional *20-second centrifuge after adding AHG* to the solution - A total centrifugation time of *200 seconds* The **Enhance (LISS)** reagent is added to the indirect test which allows for a stronger and faster reaction to occur - This *lowers the red blood cell charge* and *brings the molecules closer* together Skipping the washing step can cause an *interference problem* (not pro/postzone): - Washing removes unbound IgG, ### Direct vs Indirect Tests > [!summary]+ **Indirect Coombs Test** (Mom) > The *indirect antiglobulin test (IAT)* is an *in vitro* (performed in lab) that detects IgG antibodies within the *serum* of the mother. > - Two types of red blood cells (I and II) are used to create a *library of antigens* > - A 37 $\degree$ incubator must be used to simulate the conditions of a host body for the experiment to proceed properly --- > [!summary]+ **Direct Coombs Test** (Baby) > The *direct antiglobulin test (DAT)* is an *in vivo* (performed in a living organism) that detects > - No incubation period is required as the cells have already been growing within the baby's body > - Results indicate potential hemolytic disease of a newborn (HDN) > [!question] **Review Questions** > - What is another name for the coombs test? > - What is the Ab in the direct and indirect test? > - What is the antigen in the direct and indirect test? > - Why do you need I and II cells and which test (IAG or DAG) are they used in? > - Which test uses an incubator set at 37 degrees C and which test is the incubation done in the body? > - Which test are you using unknown serum and which test are you using unknown RBC’s? > - What could happen if you didn’t wash? > - What does enhance do? > - Why do a coombs test? Why not just put the Ab and antigen on a slide together and see if agglutination happens? > - How long do you centrifuge when washing and how long do you centrifuge after addition of anti-human globulin (anti-IgG)?