- Immune system
- Terminologies used in immunology
- Components of immune system
- Immune response
- Traditional laboratory testing methods
- Nontraditional laboratory testing methods
- Blood group antigens
- Blood group alloantibodies and autoantibodies
- Complement system and blood banking
- Historical overview of blood banking/blood transfusion service (BTS)
Immunity is defined as a resistance (defense mechanism) exhibited by host against invasion by any foreign antigen, including microorganisms. Immunology is the branch of medicine and biology concerned with immunity. Immunohematology is an integral part and plays an important role in transfusion medicine.
- Immunohematology deals with the serologic, genetic, biochemical and molecular study of antigens associated with membrane structures on the cellular constituents of blood (red blood cells [RBCs], white blood cells [WBCs], and platelets). It also deals with the immunologic properties and reactions of blood components and constituents.
- Transfusion medicine (Fig. 1.1) includes the transfusion of blood, its components, and derivatives.
Basic concepts of immunology: For understanding the principles of immunohematology, it is necessary to know the basic concepts of immunology.
These include the antigens, antibodies, antigen-antibody reactions and complement affecting the antigen-antibody reaction. The application of immunohematology principles in the clinical laboratory is usually carried out in the blood bank or transfusion services department. Immunohematologists perform and interpret various serologic and molecular assays which help in the diagnosis, prevention, and management of immunization associated with transfusion, pregnancy, and organ transplantation.
Role of Immune System
The immune system plays two main roles in the human body.
- It provides the immune mechanism which protects the body against external foreign substances. The immune response is a highly evolved system and is necessary for survival. It rapidly responds to any foreign material or pathogens that invade the body and can initiate a series of events to eliminate this foreign material or pathogens.
- It plays an important role in the identification and destruction of abnormal cells. These abnormal cells may be malignant cells, cells infected with microorganism or cells coated with antibodies. It has an ability to distinguish between self and nonself antigens.
Immune system is like a double-edged sword. Though immune system is protective in most of the situations, sometimes a hyperactive immune system may cause fatal diseases.
The immune system consists of two lines of defense namely innate and adaptive immune response/immunity.
Innate (Natural/Native) Immune Response
Its salient features are:
- First line of defense present by birth.
- Provides immediate initial protection against an invading pathogen. It is triggered by substances which indicate potential danger to a host. These substances are common to many pathogens, such as bacterial lipopolysaccharide or viral nucleic acid.
- Does not depend on the prior contact with foreign antigen or microbes.
- Lacks specificity, but highly effective.
- Triggers the adaptive immune response.
- No memory and no self/nonself recognition are seen.
- Innate immune cells: These include monocyte-derived macrophages, neutrophils (polymorphonuclear neutrophils [PMNs] and dendritic cells [DCs]).
Adaptive Immune Response
If the innate immune system fails to provide effective protection against invading microbes, the adaptive immune system is activated. Thus, it is the second and more complex immune response that may follow the innate response. It specifically targets antigens present in the immunizing substance. It is characterized by the development of antigen-specific antibody and T cell responses. Three characteristic features are: (1) specificity, (2) diversity and (3) memory. Other salient features are:
- Second line of defense acquired during life.
- Capable of recognizing both microbial and nonmicrobial substances.
- Takes more time (days to weeks) to develop and is more powerful than innate immunity.
- Long-lasting protection.
- Prior exposure to antigen is present.
- Adaptive immune cells: T and B lymphocytes, and null cells.
TERMINOLOGIES USED IN IMMUNOLOGY
Transfusion medicine needs sound knowledge of antigens and antibodies. For understanding the immune system, it is essential to know some essential terms used in immunology.
- Antigen (Ag): Any substance (usually foreign or a nonself) that is recognized as foreign by the body and capable of inducing immune response (antibody formation) in an immunocompetent individual. These antigens bind specifically to an antibody or cell-surface receptors of T lymphocytes. All antigens are not capable of eliciting an immune response. Blood group antigens are present on the surface of RBCs.
- Immunogen: It is a substance capable of provoking an antibody-mediated immune response when it is introduced into an immunocompetent host to whom it is foreign. The terms antigen and immunogen are often used synonymously. The immune response is initiated by the presentation of an antigen (initiates formation of and reacts with an antibody) or immunogen (initiates an immune response). The term antigen is more commonly used in blood banking because the primary testing is the detection of antibodies to blood group antigens.
- Immunogenicity: The ability of an antigen to elicit an immune response is known as its immunogenicity. The immunogenicity of an antigen is depends on:
- Characteristics of antigens: These include degree of foreignness, molecular size and configuration, temperature, pH, and ionic environment and antigenic complexity (depends on the number of available epitopes or antigenic determinants).
- Host's genetically determined immune responsiveness.
- Antibody (Ab): If a foreign antigen is introduced into an immunocompetent individual, a protein produced in response to it is called an antibody. Antibody is an immunoglobulin that is produced and secreted by activated B lymphocytes (plasma cells derived from B lymphocytes) after stimulation by a specific immunogen. Immunoglobulin proteins consist of two identical heavy chains and two identical light chains. The light chains recognize a particular epitope on an antigen and facilitate clearance of that antigen.While all antibodies are immunoglobulins, not all immunoglobulins are antibodies. Immunoglobulin molecules for which no complementary material or antigen has been recognized are simply called immunoglobulins, not antibodies.
- Alloantibodies: They are formed in response to antigens from individuals of the same species. These are the type of antibodies involved in transfusion reactions.
- Heteroantibodies (xenoantibodies): They are antibodies produced in response to antigens from another species.
- Autoantibodies: They are made in response to the body's own antigens.
- Antigen-presenting cell (APC): APC is any cell that can process and present antigenic peptides in association with class II major histocompatibility complex (MHC) molecules and deliver a costimulatory signal necessary for T cell activation. The professional APCs include macrophages, DCs, and B cells. Nonprofessional APCs, which function in antigen presentation only for short periods include thymic epithelial cells and vascular endothelial cells.
- Epitope (antigenic determinant): It is a portion/site of an immunogen/antigen that is recognized and combines specifically with an antibody of B lymphocyte or antigen receptor of a T lymphocyte (T-cell receptor [TCR]-MHC combination or TCR ligand-CD1 complex).
- Haptens: They are well-defined molecules that are too small to be immunogenic (i.e. they cannot stimulate antibody production) by themselves but can induce an antibody response when attached to (coupled with) a carrier protein. The hapten molecules have a molecular weight (MW) less than 10,000 daltons (D). The carrier protein should have a MW greater than 10,000 D.
- Immune system: It is a collective term for all the cells and tissues involved in immune activity (host defense system). Included in this system are the cells of the immune system, the thymus, lymph nodes, spleen, bone marrow, portions of the liver, gastrointestinal tract (GIT) and mucosa-associated lymphoid tissue.
- Receptor: It is a molecule or cell membrane protein molecule whose configuration allows it to form a tightly fitting complex with another molecule of complementary shape (ligand which is molecule that binds to a receptor).
- Cytokine: It is a low molecular weight protein secreted from an activated cell that affects the function or activity of other cells. Cytokines regulate the intensity and duration of the immune response by exerting a variety of effects on lymphocytes and other immune cells that express the appropriate receptor.
- Clone: It is a population of genetically identical cells derived from successive divisions of a single progenitor cell (a cell that originates from a stem cell and differentiates into a more specialized cell).
COMPONENTS OF IMMUNE SYSTEM
Immune system is made up of special cells, proteins, tissues and organs.
Organs Involved in Immune System
They may be central organs or peripheral organs.
- Central organs: Bone marrow, liver and thymus.
- Peripheral organs: Lymph nodes and spleen.
The mucosal-associated lymphoid tissues (i.e. GIT-associated and bronchus-associated lymphoid tissues) also play an important role by involving both central and peripheral functions.
Cells of the Immune System
Cells of immune responses (lymphocytes and other cells) migrate among lymphoid and other tissues and the vascular and lymphatic circulations.
- Lymphocytes are the primary cells involved:
- Naïve lymphocytes
- T lymphocytes
- B lymphocytes
- Natural killer (NK) cells
- Dendritic cells
These are mature lymphocytes which have not encountered the antigen (immunologically inexperienced). After the lymphocytes are activated by recognition of antigens, they differentiate into:
- Effector cells: They perform the function of eliminating microbes.
- Memory cells: They live in a state of heightened awareness and are better able to combat the microbe in case it infects again.
T (thymus-derived) lymphocytes develop from precursors in the thymus.
Distribution: Mature T cells are found in:
- Peripheral blood where it constitutes 60–70% of lymphocytes
- T cell zones of peripheral lymphoid organs namely paracortical region of lymph node and periarteriolar sheaths of spleen.
Subsets of T lymphocytes: Naïve T cells can differentiate into two major subtypes namely (1) CD4 and (2) CD8.
- T helper cells: These cells have a cell surface marker called CD4 and hence are also called CD4+ cells. They help B cells in antibody formation; constitute two-thirds of circulating T cells. They recognize antigen presented by class II human leukocyte antigen (HLA) molecules.
B (bone marrow-derived) lymphocytes develop from precursors in the bone marrow.
- Peripheral blood: Mature B cells constitute 10–20% of the circulating peripheral lymphocyte population.
- Peripheral lymphoid tissues: Lymph nodes (cortex), spleen (white pulp), and mucosa-associated lymphoid tissues (pharyngeal tonsils and Peyer's patches of GIT).
Functions of B cells: All the mature, naïve B cells express membrane-bound immunoglobulins on their surface that functions as B-cell receptors (BCRs) for antigen. B cells recognize antigen via these BCRs.
- Production of antibodies: The primary function of B cells is to produce antibodies. After stimulation by antigen and other signals, B cells develop into plasma cells. These cells secrete antibodies which are the mediators of humoral immunity.
- Antigen-presenting cell: B cells also serve as APCs and are very efficient at antigen processing.
As the name suggests these cells have numerous fine cytoplasmic processes that resemble dendrites. These are important APCs in the body.
Macrophages are a part of the mononuclear phagocyte system.
Processing of antigen: In adaptive immune response, macrophages process the antigens present in the phagocytosed microbes and protein antigens. After processing, the antigen is presented to T cells and thus, they function as APCs in T cell activation.
Adaptive Immune Responses
It can be classified into two main divisions.
- Humoral immunity: In this type, immunity is mediated by soluble protein products called antibodies produced by B lymphocytes and helper T cells. Antibody is capable of reacting with the specific antigen responsible for its production. Macrophages also participate in the effector phase of humoral immunity. Macrophages get activated by interferon-gamma (IFN-γ).
- Cell-mediated immunity (cellular immunity): Cellular immunity is mediated T lymphocytes, macrophages and their soluble products called cytokines. It is localized reaction to organism, usually intracellular pathogens. Macrophages are main effector cells in certain types of cell-mediated immunity, the reaction that serves to eliminate intracellular microbes. In this type of response, T cells activate macrophages and increase their capability to kill ingested microbes. Macrophages efficiently phagocytose and destroy microbes which are opsonized (coated) by immunoglobulin G (IgG) or C3b through their respective receptors. Cell-mediated cytotoxicity is important in lysis of virus infected cells and rejection of allograft and tumor cells. Other cytotoxic cells involved in cell-mediated immune response are natural cells (NK). These NK cells are able to attack the target cells and kill them.
Components of Adaptive Immune Response
Natural Killer Cells
- Nonphagocytic large (little larger than small lymphocytes) granular (numerous cytoplasmic azurophilic granules) lymphocytes.
- Comprise about 5–15% of human peripheral lymphoid cells.
Function: Natural killer cells provide defense against many viral infections and other intracellular pathogens and also has antitumor activity, causing lysis of cells with which they react. Killing of the cells is performed without prior exposure to or activation by these microbes or tumors. Because of this ability, NK cells act an early line of defense against viral infections and few tumors.
Major histocompatibility complex molecules (Discussed in chapter 23).
The function of the immune system is to defend the body from externally derived agents and from potentially dangerous self-constituents. The main effector cells of specific immunity are lymphocytes. The lymphocytes possess receptors capable of discriminating one antigen from another (based on differences in their molecular configuration).
Immunoglobulins are a group of serum proteins.
Antibodies: Immunoglobulins for which a corresponding antigen can be identified are called antibodies.
Immunoglobulins: They lack corresponding antigen are simply called immunoglobulins and are not antibodies.
Types of Immunoglobulins
There are five classes of immunoglobulins designated as: (1) IgM, (2) IgG, (3) IgA, (4) IgD, and (5) IgE. Of these, IgM, IgG and IgA (rarely) antibodies are produced against RBC antigens and mainly involved in blood group serology.
Structure of Immunoglobulin Molecule (Fig. 1.2)
Immunoglobulin consists of amino acid molecules linked by peptide bonds forming amino acid chains.
Heavy and light chains: All immunoglobulins share the same basic structure consisting of four chain molecules. The basic immunoglobulin unit consists of two identical heavy chains and two identical light chains held together by disulfide bonds. Immunoglobulin molecules are proteins and therefore have two terminal regions namely (1) the amino (-NH2) terminal and (2) the carboxyl (-COOH) terminal.
- Light chains: The light chains belong to two antigenetically different isotypes, i.e. (1) Kappa (K) and (2) Lambda (λ). In any immunoglobulin molecule, the two light chains are always identical, being either Kappa or Lambda.
- Heavy chains: The heavy chains are different for each class of immunoglobulins (Table 1.1).
- Disulfide bond: Each light chain is joined to one heavy chain by a disulfide bond. One or more disulfide bonds link the two heavy chains in an area of considerable flexibility called the hinge region. The four chains bound by covalent (disulfide) and noncovalent bonds give “Y” configuration.Fig. 1.2: Schematic representation of basic immunoglobulin (IgG) structure. The inset shows formation of antigen-binding fragment (Fab) and constant fragment (Fc) after enzymatic cleavage of the IgG molecule by papain.
Table 1.1 Types of heavy chain in different immunoglobulin molecules.Type of immunoglobulinType of heavy chainIgAAlpha (α)IgGGamma (Γ)IgMMu (µ)IgDDelta (δ)IgEEpsilon (ϵ)
- Variable region: The portion near the amino terminus or N-terminus of both light and heavy chains of immunoglobulins is called variable region. The antigenic specificity of the immunoglobulin molecule lies in this portion. They are termed variable because they are structured according to the great variation in antibody specificity.
- Constant portion: The carboxyl region of all heavy chains and the light chains has a relatively constant amino acid sequence and is called as the constant region. The five isotypes of heavy chains and two of light chains are determined by the amino acid sequences of the constant portion.
Digestion of Immunoglobulins
Digestion of an immunoglobulin molecule with the proteolytic enzyme papain results in cleavage or splitting of the heavy chain at the hinge region. This produces three separate fragments namely: two Fab and one Fc fragments (Fig. 1.2 inset).
- Two Fab fragments: These fragments are identical and consist of one light chain linked to the N-terminal half of the heavy chain. These N-terminal fragments retain the specificity of the antibody and are called Fab fragments (fraction antigen binding). Structurally and functionally, the Fab fragments consist of the portions of the immunoglobulin from the hinge region to the amino terminal end and are the regions responsible for binding antigen.
- One Fc fragment: The Fc fragment is that portion of the immunoglobulin molecule from the carboxyl region to the hinge region (from both heavy chains) still joined to one another by the hinge region disulfide bonds. This is the nonantibody protein fragment capable of crystallization and is called Fc fragment. Fc fragments on IgG antibody is responsible for complement fixation, for placental transfer, monocyte binding by Fc receptors on cells and reaction with antihuman globulin (AHG).
Individual Immunoglobulin Classes
IgM Antibodies (Fig. 1.3)
These antibodies readily and very strongly agglutinate the red cells carrying the corresponding antigens in saline. Therefore, they are known as complete antibodies. IgM exists in serum as a pentamer and cannot cross the placental barrier. They activate complement through the classic pathway and markedly enhance the inflammatory and phagocytic defense mechanisms. The optimal temperature is room temperature (i.e. 20–24°C).
IgG Antibodies (Fig. 1.4)
Immunoglobulin G antibodies are also called “incomplete antibody”, because they do not cause agglutination of red cells with corresponding antigen in saline. They can readily cross the placenta and responsible for hemolytic disease of newborn (HDN). It tends to combine with and remain attached to cell surface antigens, where its presence can be detected in vitro by antiglobulin testing. In vivo, cells or particles coated with IgG undergo markedly enhanced interaction with cells that have receptors for the Fc portion of gamma chains, especially neutrophils and macrophages. The optimum temperature for reaction of IgG is 37°C. IgG antibodies account for the majority of the clinically significant antibodies directed against blood group antigens.
Fig. 1.3: IgM pentamer (polymer formed from five molecules of a monomer) immunoglobulin molecule (joined together).
There are four subclasses of IgG: (1) IgG1, (2) IgG2, (3) IgG3, and (4) IgG4.
- Most IgG antibodies contain all four subclasses. However, some are predominantly or exclusively composed of a single subclass.
- The subclasses have different biologic properties.
- All bind to the crystallizable fragment (Fc) receptors on macrophages. All can cross the placental barrier. All except IgG4 are capable of binding to complement through the classic pathway.
- IgG1 and IgG3 bind complement much more efficiently than IgG2.
- IgG1 constitutes 65–70% of the total IgG found in serum.
IgA Antibodies (Fig. 1.5)
It serves no physiologic function. Most of the IgA mass and all of its physiologic significance exist in mucosal secretions. It is important to know that people deficient in IgA may have anti-IgA. IgA antibodies against RBC antigens usually occur with IgG and IgM antibodies having the same specificity. These IgA antibodies neither cross the placental barrier nor do they fix complement. IgA antibodies can cause agglutination in saline.
The location of cellular components, red cell antigen and antibody in a blood sample is depicted in Figure 1.6.
The salient features of antibodies (immunoglobulins) are presented in Table 1.2.
Immune response after exposure to an antigen is influenced by the host's previous history with the foreign material. There are two types of immune responses: (1) primary and (2) secondary.
Primary immune response: It is the response of the body when an immunocompetent individual is exposed for first time to a foreign antigen (nonself antigen). In this, there is a lag period/phase, i.e. the time between exposure to the antigen and appearance of detectable antibody. It can vary from a few days to weeks or even months. It depends on factors like nature and quantity of the antigen, route of administration and protein synthesizing capacities of the host. The antibody that appears in the blood after first contact with an antigen is always IgM type.
Fig. 1.6: Blood sample depicting the location of cellular components, red cell antigen and antibody. The serum or plasma contains the antibody, whereas the red cell membrane contains the antigen.
After some days, IgG becomes detectable. If there is no further exposure to the antigen, the level of circulating IgM antibody peaks and then declines, while IgG antibody persists for longer time. Apart from immune responses, the primary immune response generates memory cells. These memory cells contribute to the immune response on second or subsequent exposure to the same antigen (i.e. secondary or anamnestic immune response).
Anamnestic or secondary immune response: Even if the antigen that caused the primary response disappears from the body, circulating T cells and memory B cells continue to persist. When there is a subsequent or second contact with the same antigen, memory B cells respond far more rapidly than unstimulated cells. There is no lag period and the dose of the antigen can be very small. The memory cells exhibit rapid proliferation of the IgG secreting progeny. This is called anamnestic or secondary response. Within a short time (within hours or a day), the level of circulating IgG antibody rises sharply. The IgG response may be as much as 100 times greater than the primary response. The affinity of antibody molecules for the antigens will also be greater than in primary response.
Antibody level and time of development of primary and secondary antibody responses are shown in Figure 1.7.
Differences between primary and secondary immune response are presented in Table 1.3.
Red Cell Antigen-Antibody Reactions in Vivo
Transfusion, Pregnancy and Immune Response
During transfusion and pregnancy, a patient is exposed to many potentially foreign antigens present on red cells, white cells, and platelets. These foreign antigens have immunogenic potentiality. These foreign antigens may activate the immune system of the patients or patient may be “sensitized”, with the resultant production of circulating antibodies. The antibodies produced in response to transfusion and pregnancies are classified as alloantibodies.
Antibody screen test: It is performed on the recipient to detect any existing red cell alloantibodies before transfusion.
- Detection of alloantibody: If a red cell alloantibody is found, a test is done to identify the specificity of the antibody.
- Specificity of alloantibody: Once the specificity is identified, donor units lacking the red cell antigen are selected for transfusion.
- Importance: Detecting and identifying alloantibodies in the patient before transfusion are important to avoid the formation of antigen-antibody complexes in vivo (within the patient's body), which would reduce the survival of the transfused cells.
Immunization during pregnancy: Immunization may also occur during pregnancy due to entry of fetal blood cells into the maternal circulation at delivery. Alloantibody may develop as an immune response to RBC, WBC, or platelet antigens of fetal origin. Routinely females are screened during the first trimester of pregnancy for the presence of red cell alloantibodies. These red cell alloantibodies can destroy fetal red blood cells before or after delivery. The destruction of red cell may lead to clinical complications due to anemia and high levels of bilirubin in the fetus or newborn.
Red Cell Antigen-Antibody Reactions in Vitro
The combination of antibody with antigen produces a variety of observable results. Antigen-antibody reactions are important in immunohematology. In blood group serology (immunohematology), the most common reactions are as follows:
- Agglutination: Hemagglutination
Agglutination and Hemagglutination
Antigen-antibody reactions occurring in laboratory testing (in vitro) are detected by visible agglutination of the RBCs (hemagglutination) or development of hemolysis at the completion of testing (a positive result).
Hemagglutination: It produces the clumping of red cells that result when antibody molecules combine with antigenic determinants on adjacent red cells. This brings RBCs together and forms a visible aggregate.
- A positive reaction in immunohematologic testing is indicated by agglutination. A positive result indicates that an antigen-antibody immune complex was formed, and the specificity of the antibody matched the antigen in the test system.
- A negative reaction in immunohematologic testing is indicated by no agglutination. Negative result/reaction suggests that there is no formation of antigen-antibody complex and indicates that the antibody in the test system is not specific for the antigen.
Agglutination is the end point for most test involving red cells and blood group antibodies. It is the primary and most common reaction observed in routine transfusion practice.
Stages of agglutination
Agglutination occurs in two stages namely (1) sensitization and (2) visible agglutination (lattice formation).
Sensitization (or antibody binding/attachment to red cells): In the first stage of red cell agglutination, there is simple coating or binding of an antibody to an antigen on the red cell membrane. This stage needs an immunologic recognition between the antigen and antibody. During this recognition stage, the antigen-binding sites of the antibodies become closely associated with the antigenic determinants (epitopes) on the RBC membrane (Fig. 1.8). The antibodies and antigens are held together loosely by noncovalent bonds. This does not produce clumping or visible agglutination of red cells in saline. Since no visible agglutination is seen, an additional step is needed to produce visible agglutination or to otherwise measure the reaction by the use of albumin, proteolytic enzymes, or AHG reagent. This stage depends on factors such as the pH, temperature of the reaction, incubation time, and ionic strength of the suspension medium.
Visible agglutination (Lattice formation): After the red cells have been sensitized with antibody molecules, visible agglutination (Fig. 1.9) occurs when several RBCs are physically joined together by the union of antigen with antibody. This stage depends on factors such as distance between red cells, optimal concentrations of antigen and antibody, and time and speed of centrifugation.
- Enhancement of contact: The use of proteolytic enzymes (e.g. papain or ficin) can increase/enhance cell-to-cell contact of RBCs.
- Adding antiserum: RBCs sensitized by incomplete antibodies (antibodies that will not react in saline) agglutinate when antiserum against human IgG is added (antiglobulin/Coombs test).
Factors affecting agglutination
Agglutination is a reversible chemical reaction. Various factors can affect reactivity of antigen-antibody RBC agglutination reactions. These factors can be manipulated to enhance (or decrease) agglutination.
Fig. 1.9: Agglutination is the clumping of red cells together because of interactions with specific antibodies.
Agglutination reactions are affected by the concentration of the reactants (antigen and antibody) and by factors such as pH, temperature, and ionic strength. The surface charge, antibody isotype, RBC antigen dosage, and the use of various enhancement media, AHG reagents, and enzymes are all important in antigen-antibody reactions. The most important factors are discussed in the following sections:
Antigen-Antibody ratio (cell-to-serum ratio) (Fig. 1.10)
Amount of available antigen and antibody affects hemagglutination.
- Equivalence: For agglutination reactions to occur, it is necessary to have optimal proportions of antigen and antibody, i.e. equal amount/proportions of antigen-antibody. Any deviation from this ratio decreases the efficiency of the reaction and a loss of the zone of equivalence between antigen and antibody ratio. Thus, adding equal volumes of serum and 2–5% suspension of red cells is sufficient and is recommended for all routine blood banking procedures. The recommended ratio in red cell serology is one drop of serum to one drop of 2–5% red cells (or 2 drops of serum to 1 drop of the RBC suspension). It gives the proper balance between antigen and antibody to allow sensitization and agglutination to occur. This ratio may be altered, depending on the test method used.
- Prozone: It occurs when antibody molecules are in excess than that of available antigenic sites. This results in false-negative reactions. When antibody (immunoglobulin) is present in the test system in excess (when compared to antigen concentration), false-negative reactions occur as a result of prozone.
- Antigen excess (postzone): When the antigen is in excess, false-negative reactions occur due to postzone effect. In both prozone and postzone effect, the agglutination may not occur. This can give rise to false-negative results.
- The antigen-antibody test systems can be manipulated to overcome the effects of excessive antigen or antibody. If this is suspected following steps will help to correct it.
- Excessive antibody: If the problem is due to excessive antibody, the plasma or serum may be diluted with the appropriate buffer.
- Excessive antigen: The problem of excessive antigen can be solved by increasing the serum-to-cell ratio, which increases the number of antibodies available to bind with each RBC.
- Weakly reactive antibody: If the antibody is weakly reactive (weak expression of antigen on RBCs—dosage effect), increasing the antibodies present can increase the test's sensitivity.In such cases, the serum-cell-ratio may be doubled (2 drops serum and 1 drop 2–5% red cell). This provides more antibodies to react with the available antigens. While investigating adverse transfusion reactions, it may be desirable to increase the serum-cell-ratio by as much as 10- to 20-fold. This should be done only when enhancing media or potentiators have not been included in the test system.
Effect of pH
The ideal pH for antigen-antibody reactions in which most antibodies react best at a neutral pH. It ranges between 6.5 and 7.5, which is similar to the pH of normal plasma or serum. The pH values below 6 or above 8 reduce the reactivity. Stored saline has a pH of 5.0–6.0, hence buffered saline is preferred in serologic testing. However, some anti-M show enhanced reactivity at a pH of 6.5 and acidifying the test system may help in distinguishing anti-M from other antibodies.
Temperature and phase of reactivity
The optimal temperature at which an antibody reacts can provide useful clues to antibody identity. Depending on the thermal specificity, most antibodies form two broad categories:
- Those reactive at “cold” temperature (e.g. 4–25°C). IgM antibodies react best at 4–25°C.
- Those reactive at “warm” temperature (e.g. 30–37°C). IgG antibodies react best at 30–37°C.
Temperature of agglutination is determined by the nature of antigen and the type of reaction and not by the class of antibody. Binding with carbohydrate antigens (as with ABO antigen-antibody reactions) occurs best at low temperatures, while bindings with protein antigens (as with Rh antigen-antibody reaction) occurs best at 37°C due to the protein nature of the antigen. Antibodies that react in vitro only at temperature below 30°C rarely cause destruction of transfused antigen-positive red cells and are considered clinically insignificant. When performing pretransfusion compatibility testing, the focus is on clinically significant antibodies. They generally react at 37°C or with the anti-IgG in the AHG reagent.
Red cell ionic charge
Red blood cells have a negative charge at this surface which makes RBCs to repel each other. The negative charge is due to sialic acid molecules on the surface of RBCs. This natural repulsive force which holds the RBC apart is called zeta potential. This is protective and keeps RBCs from adhering to each other in the peripheral blood. The distance which keeps the RBCs apart is very small but sufficient to prevent the small IgG molecules to bridge the gap and agglutinate the red cells. However, large IgM molecules bridge the gap and bring the red cells together causing agglutination of RBCs.
In normal saline, Na+ and Cl− ions cluster around and partially neutralize opposite charges on antigens and antibody molecules. This interferes with the association of antibodies with antigen. A decrease in the ionic strength of the medium of suspension, increases the association of Ab with Ag. The use of low ionic strength solutions (LISS), or low salt media contains 0.2% sodium chloride and they decrease the ionic strength of medium.
Incubation time (length of incubation)
Incubation time is also important for antigen-antibody reactions. If incubation time is too little (less contact time), only few sensitized RBCs will be detected by routine methods. If the incubation time is allowed to continue for too long, bound antibody may begin to dissociate from the RBCs. Incubation time is different for different blood group antibodies. Incubation time also depends on temperature and medium in which the reaction takes place. Optimum incubation time for most blood group reactions in saline environment is 30–60 minutes at 37°C. Weak reactive antibodies may require longer time. Addition of enhancement agents or potentiators may shorten the incubation time. For example, enhancement agents like LISS or polyethylene glycol (PEG) can reduce the incubation time to 10–15 minutes at 37°C.
Freshness of serum and RBCs
Best antigen-antibody reaction occurs with fresh serum and freshly prepared red cells. If serum is not immediately used, it should be stored at −20°C or lower.
Agglutination reactions for IgM antibodies and their corresponding RBC antigens are easily observed in saline medium and these antibodies usually do not need enhancement or modifications to react strongly with antigens. IgG antibodies react best at 37°C and are generally responsible for hemolytic transfusion reactions and HDN. Hence, detection of IgG antibodies is clinically more significant than IgM. Many enhancement techniques or potentiators are available to discover the presence of IgG antibodies. Many of the enhancement media act by reducing the zeta potential of RBC membranes. Reducing the zeta potential allows the more positively charged antibodies to get closer to the negatively charged RBCs. Thus, enhancement techniques or potentiators increase RBC agglutination by IgG molecules. Various methods for enhancement are:
- Physical methods: Centrifugation and agitation enhance antigen-antibody reaction.
- Centrifugation: It is an effective method to enhance agglutination reactions. It reduces the reaction time by increasing the gravitational forces on the reactants, brings the cells close together and increases the chance of association between antibody and antigen. During centrifugation, sensitized RBCs overcome their natural repulsive effect (zeta potential) for each other and agglutinate more efficiently. High-speed centrifugation is one of the most efficient methods used in blood banking. However, centrifugation should not cause packing of cells too tightly, which may lead to false-positive reactions.
- Agitation: Another method of enhancing antigen-antibody reaction often employed by the shakers used for rapid plasma reagin (RPR) and Western blot testing.
- Chemical methods:
- Bovine albumin (22% or 30% concentration): It increases the dielectric constant of the medium and reduces the zeta potential (i.e. reduce electric repulsion between cells). It also affects the surface tension between cells, thus causing antibody-coated cells to agglutinate. Thus, by reducing zeta potential and surface tension, albumin enhances antigen-antibody binding. Bovine albumin does not cause agglutination of noncoated cells.
- Enzymes: Papain is the proteolytic enzyme most commonly used in blood group serology. Others include bromelin, ficin, and trypsin. These enzymes act by:
- Lowering the zeta potential: Sialic acid is the major contributor of the net negative change at the red cell surface which keeps cells separated from each other in an ionic suspending medium. Enzymes cleave sialic acid molecules and reduce the negative charge on RBCs.
- Cleaving of protein also increases the surface tension between cells thus predisposing to agglutination.
- They cause spicule formation on the red cell. This increases the potential number of contact points.Note: Certain red cell antigens can undergo denaturation by enzyme treatment, e.g. M, N, S, Fya, and Fyb. Thus, it is important not to use enzyme treatment while detecting any of these antigens.
- Antihuman globulin (AHG) reagent: AHG causes agglutination of sensitized/coated cells. AHG bridges the gap between the IgG molecules attached to the red cells and enhances their agglutination. This is the most common as well most sensitive method used to detect antigen-antibody reaction in immunohematology.
The direct AHG test is used to determine if RBCs are coated with antibody or complement or both.
Polyethylene glycol and polybrene are macromolecule additives used with LISS to enhance agglutination reactions. PEG is more effective than albumin, LISS, or polybrene for detection of weak antibodies. These reagents have been used in automated and manual testing systems.
Grading and scoring of hemagglutination are presented in Table 1.4.
In immunohematology laboratory, apart from agglutination as an indicator of an antigen-antibody reaction, red cell hemolysis in the tube is also an indicator of the activity of an antigen and antibody (antigen-antibody reaction) in vitro.
- Hemolysis (Fig. 1.11) is the rupture or breakdown of red cells with release of intracellular hemoglobin. In vitro, hemolysis requires activation of complement cascade. Complement system gets activated when there is antigen-antibody complex (immune complex). Hemolysis does not occur if the antigen-antibody reaction takes place in serum that lacks complement (stored blood) or in anticoagulant that chelates/binds to Ca++ and Mg++ (both Ca++ and Mg++ necessary for complement activation). Hence, for demonstration of hemolysis fresh serum samples (without anticoagulants) should be used.
Table 1.4 Grading and scoring of hemagglutination.GradeAppearanceScore4+Red cell button—one solid aggregate with a clear background103+Several medium to large aggregates with a clear background82+Many small to medium aggregates with a clear background51+Many small aggregates with a turbid background with many free red cells3+ or wFew small aggregates with many unagglutinated cells2±m or +mAggregates visible only under microscopic examination10Negative—absence of aggregates (no agglutination)0RRouleaux (nonspecific aggregation that appears like a stack of coins and disappears with addition of saline)NAHHemolysis—presence of free hemoglobin in the serum10
- Hemolysin: Antibodies that have the capacity to activate complement on reacting with antigens on red cells and cause hemolysis are called hemolysins.
- Many blood group antibodies on reacting with the antigens on red cells activate the complement and produce membrane attack complex (MAC). MAC causes damage to RBC membrane leading to destruction of RBCs. This in turn releases the intracellular fluid in the RBCs into the serum.
- In the test system, hemolysis is identified by the pink or red coloration of the supernatant fluid after tubes are centrifuged. IgM antibodies predominantly activate complement while IgG rarely does so.
- Some red cell antibodies characteristically produce hemolysis in vitro, such as antibodies to the Lewis system antigens and anti-Vel.
Note: Testing for hemolysin is mandatory prior to release of the so-called universal red cells/whole blood of group “O” to a recipient with non-O (A, B, AB) group.
Soluble form of blood group antigens can also combine with soluble blood group antibody. It will result in full or partial neutralization (inhibition) of antibody. There is no formation of a visible precipitate. In this reaction, if the strength of the antibody diminishes or if it disappears completely, an antigen-antibody reaction can be assumed to have taken place.
Missing or weak antigens/antibodies can often be detected with the help of neutralization reactions.
Precipitation (Fig. 1.12)
When soluble antibody reacts with soluble antigen and forms an insoluble, usually visible complex, the reaction is called as precipitation. Such complexes are seen in test tubes as a sediment or ring and in agar gels as a white line. Precipitation is the end point of procedures such as immunodiffusion and immunoelectrophoresis. Examples for precipitation reactions are venereal disease research laboratory (VDRL) and RPR tests performed in the blood bank to screen for syphilis.
- Antigen and antibody should be present in optimal proportions for the occurrence of precipitation. In an ideal reactive condition, an equivalent amount of antigen and antibody binds.
- Prozone phenomenon (refer pages 12 and 13): If there is excess of unbound Ab (immunoglobulin), there will be very few Ag sites to combine with the molecules and the lattice structure is not formed. Ag-Ab complexes are formed but do not accumulate sufficiently to form a visible lattice. An excess of leads of antibody leads to a phenomenon called a prozone. It is important to rule out prozone phenomenon while screening for atypical antibodies like anti-D in a woman giving a sample for indirect antiglobulin test, during antenatal checkup.
- A surplus of antigen leads to a postzone effect (refer page 13).
TRADITIONAL LABORATORY TESTING METHODS
These include hemagglutination (a special type of agglutination), precipitation, agglutination inhibition, and hemolysis.
Other techniques which are used to quantify antigen or antibody with the use of a radioisotope, enzyme, or fluorescent label—such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA), Western blotting (WB), and immunofluorescence (IF).
In transfusion medicine, much laboratory testing involves detection and identification of antibodies in patient's plasma. These assays can be mainly divided into: (1) fluid-phase assays (agglutination-based methods) and (2) solid-phase assays.
Fluid-Phase Assays (Agglutination-Based Methods)
Uses of Agglutination-Based Tests in Blood Bank
Agglutination is used for:
- Serologic cross-matching (donor RBCs incubated with recipient plasma or serum)
- Screening for unexpected antibodies (reagent RBCs of known blood group antigen composition incubated with recipient plasma or serum)
- Blood group antigen phenotyping of the donor or recipient (test RBCs are incubated with monoclonal antibodies or reagent-quality antisera of known specificity).
Methods (Refer Pages 71-84 of Chapter 4)
- Manual tube testing: Agglutination detected by adhesion of RBCs to one another in post-centrifuge pellet.
- Microtiter plate: Agglutination visualized by spread pattern of RBCs in individual wells.
- Column agglutination technique:
- Gel-based testing: After agglutination is allowed to take place, the reaction mixture is centrifuged through a gel-matrix (usually composed of dextran-acrylamide). Unagglutinated RBCs pass through the gel, whereas larger, agglutinated RBCs are retained at the top or within the matrix. Details are presented in Chapter 4.
- Glass microbeads technology (refer pages 78-82)
Agglutination reaction tests are sensitive and easy to perform. However, the formation of agglutination depends on antigen-antibody ratio (refer Fig. 1.10 on page 13).
In these assays, a specific antigen or antibody is immobilized on a solid matrix, usually made of plastic. It can be used for either antigen detection or antibody detection. A solution containing antigen/antibody (depends on which has to be tested, i.e. if antigen in the testing sample to be tested then antibody and vice versa) is placed on the well; polystyrene (or other plastic used) directly absorbs antigen/antibody from the solution and irreversibly binds to antigen/antibody to the plastic. The well is washed, the analyte is added and incubated with the antigen/antibody-coated solid phase, and its adherence is measured.
Solid-Phase Assays for Phenotyping RBCs (Fig. 1.13)
Antibodies specific for a known blood group antigen are coated onto the round bottom of the microtiter plates.
RBCs to be analyzed are added to the microplate wells, allowed to adhere and then the microplate is centrifuged.
- Positive reaction: Specific binding of RBC to the antibodies results in dispersion of the RBCs over the surface of the entire well. This indicated the presence of antigen on the RBCs.
- Negative reaction: No binding of RBC antigens to the antibodies on the well of microtiter plates. The red cells cluster together as a “button” at the bottom of well.
Solid-Phase Assays for Detecting Antibodies to RBC Antigens (Fig. 1.14)
In this test, antigen-coated RBCs (or red cell fragments) are coated onto the microtiter plate wells. Patient's serum to be tested is added. It is incubated and washed. If the patient serum contains antigen-specific antibodies, they will bind to antigen-coated red cells in the microtiter plate. Then indicator red cells (coated with antihuman IgG) are added.
- Positive reaction: It is characterized by diffuse adherence of the indicator red cells to the well.
- Negative reaction: It is characterized by clustering of indicator red cells in a button.
Enzyme-Linked Immunosorbent Assay
Discussed on pages 359-63 of Chapter 16.
NONTRADITIONAL LABORATORY TESTING METHODS
Discussed on pages 364-5 of Chapter 16.
Recent techniques to study immunologic reactions are fluorescence-assisted cell sorting (FACS) and flow cytometry. Flow cytometry has revolutionized the analysis of cell populations.
Principle: In flow cytometry, antibodies tagged with a fluorescent dye (i.e. fluorescent tag-labeled antibodies) against cell surface molecules are used. These antibodies are incubated with target population of cells. These “stained” cells are then passed through a flow cytometer. As the cells coated with fluorescent-labeled antibody travel in the flow cytometer, the cells are exposed to lasers which excite the fluorescent tags. This causes emission of a brightly fluorescent color of a specific wavelength that can be detected by sensors in the flow cytometer. Fluorescence is assessed on cell-by-cell basis. This allows visualization and quantification of minor population cells present in a complex mixture.
Suspension Array Technology
Suspension array technology (SAT) combines the specificity of solid-state antigen/antibody interaction (ELISA) with the sensitivity of flow cytometry. In transfusion medicine, it is used for identification of HLA-specific alloantibodies for screening platelet donors and blood group genotyping.
BLOOD GROUP ANTIGENS
The term blood group is not only used for genetically encoded red cell antigens but also to the immunologic diversity expressed by other blood constituents, including leukocytes, platelets, and plasma.
Location of gene and mode of inheritance: Most blood group genes (few exceptions) are located on the autosomal chromosomes and their inheritance follows Mendelian laws of inheritance. A majority of blood group alleles also demonstrate codominance. In codominance, the products of both alleles of a gene pair exert an observable effect and are thus equally dominant (e.g. the alleles A and B of the blood group system ABO; O is recessive to A and B). That means genetic heterozygotes at a particular locus will express both gene.
Many membrane-associated structures on blood cells act as antigens because they are capable of reacting with a complementary antibody or cell receptor. A majority of these antigens are capable of eliciting an antibody-mediated immunologic response and are thus are immunogenic (refer page 3). Each antigen can have a variety of different epitopes or specific antigenic determinants page 3. Epitopes are discrete, immunologically active regions of the antigen. The epitopes can interact with specific lymphocyte membrane receptors or secreted complementary antibody.
About a dozen of antigen systems are significant and are commonly important in the transfusion medicine. Individuals who lack certain antigens, when exposed to them may form antibodies. These antibodies may be detected on routine testing in the blood bank products.
An antigen capable of eliciting an immune response is called as immunogenicity. Blood group antigens greatly vary in their capability to elicit an immune response. The most immunogenic are A, B, and RhD antigens. Hence, all blood to be transfused must be matched for these antigens between the blood donor and the recipient. About 50–75% of D-negative individuals would produce anti-D if transfused with only one unit of D-positive blood. Apart from AB and D antigen, K followed by Fya antigens are also immunogenic.
BLOOD GROUP ALLOANTIBODIES AND AUTOANTIBODIES
Majority of clinically significant blood group antibodies are IgG or IgM type and occasionally an IgA type.
Classification of Blood Group Antibodies
Blood group antibodies can be classified as:
Alloantibody: This reacts with a foreign antigen not present on the patient's own RBC. Identification of alloantibodies and selection of compatible blood components are the most important functions of a transfusion medicine service.
- Naturally occurring antibodies: These antibodies that are present in our body in the absence of an apparent stimulus. Some alloantibodies to RBC antigens are called naturally occurring. The antigenic stimulus for this is unknown and these antibodies may appear regularly in the serum of persons who lack the corresponding antigen. For example in the ABO blood group system. These antibodies are commonly of IgM type and occur in serum without any specific red cell antigenic stimulus (e.g. anti-A, anti-B, anti-P).These antibodies are present in individuals who lack that particular antigen. They develop in infancy by 4–8 months and are maintained with little variation throughout the life. They again reduce in old age. Other naturally occurring antibodies are produced only in a small number of individuals.
- Acquired antibodies: Most blood group alloantibodies are produced as the result of immunization to foreign RBC antigens. The immunization may occur either during previous transfusion of blood components or following pregnancy. These antibodies are usually IgG type. Examples include Rh antibodies like anti-D and anti-Kell produced by external sensitization.
Autoantibody: It reacts with an antigen on the patient's own cells.
COMPLEMENT SYSTEM AND BLOOD BANKING
The complement system or complement, is a complex group of over 20 circulating serum and cell membrane proteins that play a number of biologic roles. They play most important role in immunohematology in that they are able to lyse the cell membranes of antibody-coated RBCs. They have a multiple function within the immune response. Their primary roles include immune adherence, phagocytosis, direct lysis of cells and bacteria, as well as assisting with opsonization to facilitate phagocytosis. Their peptide fragment split products play roles in inflammatory responses such as increased vascular permeability, smooth muscle contraction, chemotaxis, migration, and adherence. It is often involved in blood group reactions and immunological disorders. Complement plays an important role in the sensitization and destruction of transfused RBCs by alloantibody or the destruction of autologous RBCs by autoantibody. Complement is also important in immunohematologic testing. The complement components are unstable and heat liable. Hence, it is important for serum specimens to be fresh for blood bank testing.
The complement system is a group of plasma proteins synthesized in the liver, and are native precursor components. They are sequentially numbered from C1 to C9. The number refers to their discovery date, not to their activation sequence. The four unique serum proteins of the alternative pathway are designated by letters: factor B, factor D, factor P (properdin), and IF (initiating factor). Complement components circulate in inactive form as proenzymes, with the exception of factor D of the alternate pathway. The cleavage products of complement proteins are distinguished from parent molecules by adding suffixes from “a” to “e” as they are cleaved (e.g. C3a and C3b).
Pathways of Complement System Activation (Fig. 1.15)
The complement proteins may be activated in a cascade of events. The decisive step in complement activation is the proteolysis of the third component, C3. Cleavage of C3 can occur by any one of the three pathways: (1) the classical, (2) alternative, and (3) lectin pathways.
- Classical pathway: It is activated by antigen-antibody (Ag-Ab) complexes. The antibodies involved are IgM, IgG1, or IgG3 antibody and get activated when the C1 component binds to the Fc portion (refer page 6) of the antibody molecule.
- Alternative pathway or properdin system: It is triggered by microbial surface molecules (e.g. endotoxin, or lipopolysaccharides/LPS), complex polysaccharides, cobra venom, and other substances and does not require specific antibody for activation. Thus, they get activated in the absence of antibody.
- Lectin pathway: It directly activates C1 when plasma mannose-binding lectin (MBL) binds to mannose on microbes. MBL in turn activates proteins of the classical pathway.
Role of Complement in RBC Destruction
The reactions that take place from C5 to C9 are termed the membrane attack complex and result in lesions on the RBC surface. These lesions allow the rapid passage of ions, and the cell lyses from osmotic pressure changes. When antibody binds to intrinsic (self) RBC antigens on the RBC membrane, it activates the complement by classic pathway.
Fig. 1.15: Different pathways of activation and functions of the complement system. All pathways of activation lead to cleavage of C3.
Complement may also be activated on RBCs when an exogenous antigen (nonself, e.g. drugs like penicillin which acts as hapten) adsorbed to its cell surface. For example, penicillin-coated RBCs and forms antipenicillin antibodies.
RBC-antibody complexes usually activate complement by the classical pathway. Antibody-coated RBCs are removed by cells of the mononuclear phagocyte system.
Intravascular Hemolysis (Fig. 1.16)
Intravascular RBC hemolysis is usually caused by antibodies directed against the ABO antigens. Rarely, hemolysis may be due to other IgM blood group antibodies or some complement-fixing IgG antibodies (e.g. anti-Kidd antibodies). Intravascular lysis occurs when large amounts of complement are rapidly activated. It results in complete activation of complement cascade and generation of the terminal membrane attack complex (C5–9). This complex polymerizes to form pores in the RBC membrane and the extracellular fluid enters the cell. The RBCs swell and burst by osmotic lysis.
Majority of extravascular hemolysis is due to IgG antibodies against RBC antigen. When IgG antibodies bind RBCs, it activates the complement and the complement-coated RBCs are removed from the circulation and are destroyed in the RE (reticuloendothelial) system.
Historical Overview of Blood Banking/Blood Transfusion Service (BTS)
- The first attempt for blood transfusion was made in 1492. During this, to save life of a Pope Innocent VIII (who was in coma), an attempt was made by orally administering blood from 3 healthy boys. However, it resulted in death of all of them.
- In 1628, English physician William Harvey discovered the circulation of blood.
- During 1665–1667, Dr Richard Lower (England) and Dr Jean-Baptiste Denys, an eminent physician of King Louis XIV of France recorded successful blood transfusion in animals and reported transfusions from lambs to humans. After this, transfusing the blood from animals to humans was prohibited by law, delaying the advances in transfusion medicine for about 150 years.
- In 1795, in Philadelphia, an American Physician, Philip Syng Physick, performed the first human blood transfusion, although he does not publish this information.
- In 1818, James Blundell, a British obstetrician transfused human blood to a female with postpartum hemorrhage. During 1825–1830, he performed ten transfusions, of which five were beneficial to the patients.
- In 1840, Samuel Armstrong Lane and Blundell, undertook first successful whole blood transfusion to treat a case of hemophilia.
- Clotting was the main obstacle for transfusion of blood. In 1869, Braxton Hicks recommended the use of sodium phosphate as a nontoxic anticoagulant.
- Karl Landsteiner (Australian physician) discovered the major milestone by identifying ABO group in 1901. He also explained the rational for blood incompatibility and hemolytic transfusion reaction. Landsteiner won the Nobel Prize for Medicine for this discovery in 1930. The discovery of the ABO blood group system marked the beginning of modern blood banking and transfusion medicine. Landsteiner noted the presence of agglutinating antibodies in the serum of individuals who lacked the corresponding ABO antigen.
- AB blood group was discovered in the year 1902 by A Decastello and A Sturli.
- In 1907, Hektoen suggested that the safety of transfusion can be improved by cross-matching.
- In 1912, Roger Lee (physician) from Massachusetts General Hospital coined the term “Universal Donor” and “Universal Recipient”.
- The Rh blood group system was discovered by Karl Landsteiner, Alexander Wiener, Philip Levine and RE Stetson in 1940.
- Successful blood transfusion was achieved in 1914. Huston reported the use of sodium citrate and glucose as diluents and anticoagulant solution for transfusion.
- In 1915, sodium citrate was used by Richard Lewisohn to prevent clotting of blood. This has helped the process of collection of blood from donors and storage easier.
- In 1916, Rous and Turner introduced citrate-dextrose solution as anticoagulant for blood collection. This has resulted in more practical and safer transfusion of blood.
- MNS system was discovered in the year 1927. P blood group system was also discovered in the same year.
- Early in 1932, the first blood bank was established in Leningrad Hospital, Russia to combat blood loss in World War II.
- Dr Charles Drew first described the techniques in blood transfusion and establishment of blood bank.
- In 1930–1940 another major blood group Rh blood group system was discovered by Karl Landsteiner.
- In 1940, Edwin Cohn developed fractionation—plasma, albumin, protein.
- In 1943, P Beeson first published the transfusion-transmitted hepatitis.
- In 1945, Coombs, Mourant, and Race described the use of AHG (Coombs test) for detection of incomplete antibodies.
- Kell blood group was discovered in the year 1946. This blood group was named after Mrs Keller, the mother of first child to be affected with HDN. Kidd blood group was named after Mrs Kidd whose serum contained the antibody and antigen was named “JK” after woman's child John Kidd, who suffered HDN.
- In 1948, developed the plastic bag for blood collection.
- Duffy group was detected in 1950. Mr Duffy was a hemophilia patient.
- Bombay blood group was discovered by Dr YM Bhende and colleagues in the year 1952.
- In 1962, antihemophilic factor was discovered and use of component was understood.
- In 1964, plasmapheresis was introduced.
- In 1965, cryoprecipitate was first used.
- In 1971, HBsAg antigen detection test was introduced for safe blood transfusion.
- In 1981, reported the first case of acquired immunodeficiency syndrome (AIDS). The causative agent for AIDS was identified on 1983. In 1985, ELISA screening test for HIV antibodies was available.
- In 1985, donated blood screening for HIV was started.
- In 1998, it was made mandatory to test for hepatitis C for blood transfusion.