Notes on serology...
INTRODUCTION TO IMMUNOHEMATOLOGY DEFINITIONS
Immunity: Protection against infection or disease caused by foreign particles
Immunology: Study of the immune system and the immune response; study of antigen-antibody reactions in vivo
Serology: Study of antigen-antibody reactions in vitro
Hematology: Study of the cellular components of the blood
Immunohematology (Blood Banking): The detection, identification, and/or quantitation of antibodies involved primarily with red cells, although white cells and platelets may also be involved
TYPES OF IMMUNOHEMATOLOGY FACILITIES Transfusion Service
antibody detection and identification
compatibility testing (crossmatching)
blood component therapy (hemotherapy)
transfusion reaction workups
autoimmune hemolytic anemia workups
hemolytic disease of the newborn (HDN) workups
determining Rh immune globulin eligibility
Donor Center (Blood Center)
testing (typing, infectious disease screening)
blood component preparation
may provide reference lab services
may store rare donor blood
NATURE OF BLOOD BANKING: Blood banking involves less automation than Hematology and Clinical Chemistry. The results are not numerical other than grading reactions as 1+, 2+. 3+ and 4+. There is a heavy emphasis on the thought processes of logic, interpretation, decision making and problem solving. You need to think about what is happening in the test tube and whether the patient's red cell antigens or antibodies are involved. Blood banking can be very rewarding since there are visible life-saving aspects of providing the patient with the needed blood components. Unfortunately it is also prone to human error and the consequences of error can be fatal. There are not many places in the clinical laboratory where a patient may die from an erroneous result, but giving A or B blood to an O individual can do that. BLOOD BANK ANTIGENS AND ANTIBODIES Antigen Antigens are defined as substances recognized by the body as foreign, causing the body to produce an antibody to react specifically with it Characteristics of antigens: In order to be an antigen to you it must be foreign (not found in the host)!
Autologous antigens are your own antigens (not foreign to you)
Homologous, or allogenic, antigens are antigens from someone else (within the same species) that are foreign to you
Antigens must be chemically complex.
Proteins and polysaccharides are antigenic due to their complexity. On the other hand, lipids are antigenic only if coupled to protein or sugar.
Besides being chemically complex, antigens must also be large enough to stimulate antibody production. Their molecular weight needs to be at least 10,000.
Due to the complexity of these molecules there are specific antigenic determinant sites, or epitopes, which are those portions of the antigen that reacts specifically with the antibody.
Factors determining whether an antigen will stimulate an antibody response:
Degree of foreignness. Only human blood is transfused to humans.
Size and complexity. Although red cells are smaller than white blood cells, they tend to be more antigenic due to the complexity of the antigens on the cell surface. Some are proteins and others are oligosaccharides.
Dose of antigen administered. How much antigen is the individual exposed to and what is the frequency of that exposure.
Genetic makeup of host may also dictate whether an antibody is produced. Some individuals have a greater ability to make antibody and others have the antigen so they would not make the antibody.
Blood group antigens:
There are over 300 known blood group antigens
Over 1,000,000 different antigen sites on each red blood cell.
These antigens are attached to proteins or lipids on the red cell membrane and are usually complex sugar groups.
Some stick out far on the red cell membrane and some are buried within crypts on the membrane surface.
Antibodies Proteins produced by lymphocytes as a result of stimulation by an antigen which can then interact specifically with that particular antigen. Serum components
Human serum can be separated into albumin and globulin components
Globulins can be separated into several different parts: a. Alpha 1 and alpha 2 globulins b. Beta globulins (serum complement) c. Gamma globulins (immunoglobulins or antibodies)
Parts of an antibody: 1. Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains 2. Light chains - made of kappa or lambda chains 3. Disulfide bonds - hold chains together 4. Hinge region - allows antibody to flex to reach more antigen sites 5. Fab fragments - contains variable portion of antibody: antigen-binding sites 6. Fc fragment - contains constant portion of antibody; also site of complement activation
Classes of antibodies: 1. IgG - provides long-term immunity or protection 2. IgM - first antibody produced in response to an antigenic stimulus 3. IgA - found in secretions. Protects against infections in urinary, GI, and respiratory tracts 4. IgE - involved in allergic reactions 5. IgD - not much known about it. Surface receptor of B lymphocytes 6. Most important classes of antibodies in blood banking are IgM and IgG
Characteristics of IgG and IgM antibodies 1. Clinical significance Clinical of red cell antibodies in blood bank depend on whether they can cause in
vivo hemolysis, which in turn will cause transfusion reactions or HDN IgG will frequently cause in vivo hemolysis due to antibody coating red blood cells. IgM, with a few important exceptions, usually does NOT cause in vivo hemolysis.
The most important of these exceptions are ABO antibodies. 2. Size of the antibodies IgG is relatively small, it is comprised of only 1 immunoglobulin subunit (monomer) IgM is relatively large, it is comprised of 5 immunoglobulin subunits. (pentamer)
3. Serum concentration IgG is found in the largest concentration of all immunoglobulins in the plasma. IgM is found in relatively small amounts IgG > IgA > IgM
4. Complement activation IgG = will do it if conditions are optimal IgM = very good complement activator
5. Placental transfer IgG is small enough to cross placenta and is the only immunoglobulin capable. IgM and the other classes do not cross placenta
6. Optimum temperature of reactivity a. IgG = 37oC b. IgM = 4 oC (may react at any temperature below 30C)
7. Number of antigen-binding sites IgG has 2 binding sites IgM has 10 binding sites
Terms used to describe antibodies Immunoglobulin: antibody formed as a result of immune stimulus (exposure to foreign antigen) Naturally occurring antibody formed without prior exposure to foreign antigen Autoantibody: antibody formed to one's own antigens (abnormal condition) Alloantibody (unexpected, irregular, atypical): antibody formed to foreign antigens, but within the same species Agglutinin: antibody capable of causing agglutination when react with corresponding antigen Isoagglutinin: name commonly given to blood group antibodies anti-A and anti-B Saline agglutinin: antibody capable of causing direct agglutination of antigens suspended in a saline medium without requiring any enhancement techniques Hemolysin: antibody capable of causing hemolysis when reacting with corresponding antigen Cold antibody (cold agglutinin): antibody whose optimal temperature of reactivity is less than 30oC Warm antibody: antibody whose optimal temperature of reactivity is greater than 35oC
Monoclonal antibodies Monoclonal antibodies react with very specific antigenic determinants and therefore shows no cross-reactivity. They are not produced in humans or animals, but harvested from cells in cells grown in tissue culture. The tissue culture cells made from fusion of a plasma cell, which is the antibody producer and the myeloma cell, which provides longevity and ability to make large amounts of antibody
Monoclonal antibodies used in most reagent antisera today because contain high concentrations of highly specific antibodies and lack infectious disease hazards associated with human-source antiserum.
ANTIGEN-ANTIBODY REACTIONS IN GENERAL Rules of Thumb For in vivo Antigen-Antibody Reactions 1. If a person's cell have the antigen, the antibody should NOT be present in that person's serum 2. If an antibody to a blood group antigen is present in the serum of a person, his or her cells should lack that antigen 3. The antigens are on the cells and the antibodies are in the serum Stages of Antigen-Antibody Interaction The first stage is sensitization. Sensitization occurs when antibodies react with antigens on the cells and coat the cells.
The second stage of the reaction is agglutination. Agglutination occurs when antibodies on coated cells form cross-linkages between cells resulting in visible clumping.
FACTORS AFFECTING SENSITIZATION (In vivo or in vitro) 1. Specificity depends on the spatial and chemical "fit" between antigen and antibody
2. Since the immunoglobulins and the red cell membranes both have an electrical charge, there is an optimum pH. pH differences cause differences in chemical structures of antigens/antibodies, affecting the "fit".
3. The optimum temperature depends on the type of antibody involved. IgG antibodies react best at 37oC; IgM react best at 4oC.
4. Optimum incubation time: you need to incubate long enough to reach equilibrium, but not too long
5. The antigen's accessibility is also important since the antibodies must be able to reach antigens. Those antigens, like the ABO antigens, are on the surface of the red cell while others may be hidden in the crypts of the cell membrane.
FACTORS AFFECTING AGGLUTINATION IN VITRO Number of Antigen Sites The number of antigen sites on the red cell is important since the more antigen sites result in more antibodies being attached and forming cross-linkages. These cross-linkages result in agglutination Size and Structure of the Antibody The larger antibodies (IgM) can reach between more antigen sites on different red cells and therefore causing stronger agglutination reactions. IgM antibodies also have more binding sites to react with antigens and potentially causing cross-linkages between 5 different cells. Distance between Cells Centrifugation of the cells attempts to bring the red blood cells closer together, but even then the smaller IgG antibodies usually cannot reach between two cells. The larger antibodies, IgM, can reach between cells that are further apart and cause agglutination.
The concept Zeta potential is important to understand why the cells will maintain a certain distance from each other. Zeta potential refers to the repulsion between the red blood cells. It is due to an electric charge surrounding cells suspended in saline.
It is cause by sialic acid groups on the red blood cell membrane which gives the cells a negative charge.
The positive ions in saline attracted to the negatively charged red blood cells.
The net positive charge surrounding cells in saline keeps them far apart due to repulsion from electric charges
Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists
To overcome zeta potential techniques need to neutralize these charges One of the common techniques is: 1) Add albumin to test mixture 2) OH- groups of albumin neutralize positive charge
Antigen-Antibody Ratio The optimum ratio is 80 parts antibody to 1 part antigen. There are specific terms for variations in this ratio. Prozone - antibody excess: Antibodies saturating all antigen sites; no antibodies forming cross-linkages between cells; no agglutination Zone of equivalence: antibodies and antigens present in optimum ratio, agglutination formed Zone of antigen excess (Post-zone): too many antigens - any agglutination is hidden by masses of unagglutinated antigens
In order to get optimum antigen-antiboy concentration washed 3% saline suspension of red cells to mix with our reagents.
LABORATORY TECHNIQUES: REAGENTS, REACTION GRADING; CELL WASHING; SAMPLE COLLECTION BLOOD BANKING REAGENTS The techniques used involves mixing antigens, usually on red blood cells with antibodies. The environment where this reaction occurs can range in temperature from 4 oC to 37oC. With the most common being room temperature for ABO and the initial Rh(D) testing and 37oC when screening and identifying other clinically significant antigen-antibody reactions. In a number of situations we are looking for particular antigens on the red cell such as looking for A or B antigens to determine a patient's ABO type. Other times we may be looking for particular antibodies that may cause transfusion reactions or HDN. Depending on whether we are looking for a particular antigen or antibody will determine what reagents we are going to use. If we are looking for an A antigen on a patient's red cells, we will use known anti-A reagent that will cause agglutination of the A antigens on the red cells. If the patient has on B antigens or no ABO antigens, as in the case of an O individual, their cells will not agglutinate with anti-A reagent. Sources of Antigen Testing: The red cells may either reagent red cells with known antigens, patient red cells, or donor red cells. The reagent red cells are commercially prepared and have all the red cell antigens identified. When we use red cells where the antigens have already been determined, we can identify the possible antibodies present. The reagent cells used for blood banking include the following:
A1 and B cells for confirmation of the ABO type in all patients and donors other than newborn babies
Antibody screening cells are O cells that have been studied to determine the presence of a number of antigens for specific antibodies that are known to cause transfusion reactions and hemolytic disease of the newborn. The antibody screening technique is part of all compatibility tests done before blood is transfused. Some of the more common antibodies detected are anti-D, anti-E, anti-K.
Antibody identification cell panel are again O cells with the specific antigens known. Usually there are between 8 and 12 different cells in a cell panel. The pattern of positive and negative reactions help identify the antibody.
Sources of Antibody for Testing Antibody is found in serum. If it is the patient's serum that is being tested, we do not know what antibody may be present so we are using one of the 3 types of reagent cells listed about. If the serum is commercial reagent, the specific antibody present is already known. The commercial serum reagent is referred to as antisera. Therefore, we use Anti-A antisera to determine if a patient or donor is Type A. If we are trying to determine if the patient is Rh+ or Rh-, we will use anti-Rh (D) antisera. Known Source with known
Unknown components –
The source is either the patient or the donor
Reagent Red Blood Cells
Patient or Donor red blood cells
Patient or Donor serum or plasma
Antigen typing from other blood group systems such as Rh antigens other than D, Kell, Kidd, and Duffy
Antibody screening for antibodies form to blood group antigens other than A and B
Antibody identification to determine the specific antibodies detected in the antibody screening
Crossmatch, or compatibility testing, which determines whether donor blood can probably be safely transfused to the recipient
Source of Antigen
Detects A, B, and D
Detects antigens of Antigen typing
other blood group
Source of Antibody Commercial anti-A, anti-B, and anti-D Commercial antisera to the
Patient's RBC's or
systems (examples: K, E, Donor RBC's
(examples: anti-K, anti-E,
C, Fya, Jka)
anti-C, anti-Fya, anti-Jka)
Detects antibodies with Antibody screening specificity of RBC antigens
Commercial Screening Cells
Identifies the specificity Commercial Panel
of RBC antibodies
Determine compatible Crossmatch
in donor and patient before transfusion
GRADING REACTIONS Grading agglutination reactions gives an indication of the relative amount of antigen or antibody present. All tubes tests should be graded. The technique used in the resuspension of the cells will affect the grading of the reaction.
1. Resuspension Procedure: read only one tube at a time hold tube upright very gently shake the tube and observe how
the cells come off the cell button
2. Grading Reactions swirling off = negative coming off in chunks = positive continue shaking till all cells resuspended tilt tube, read and grade reaction
3. Grading system: 4+ = solid clump 3+ = several large clumps 2+ = small to medium sized clumps; clear background 1+ = small clumps; cloudy background +w = tiny aggregates; cloudy background + micro = positive upon microscopic examination only MF = mixed field. Small clumps amidst many unagglutinated cells. hem = hemolyzed (a positive reaction) 0 = negative, no agglutination (Never use - for negative)
WASHED CELL SUSPENSIONS 3% Red Blood Cell Concentration in Saline Between 2-5% cell suspension provides optimum antigen concentration for the tube method for red blood cells typing. Washing Red Blood Cells Before Making the 3% Suspension The purpose of washing the red blood cells is to remove plasma, which contains substance that may interfere with antigen-antibody reaction. The following may be in the plasma and may interfere with testing:
Soluble antigens such as A and B may be present and neutralize reagent.
Interfering proteins such as Wharton's jelly that is seen in newborn cord blood, cold-acting autoimmune antibodies, and increased levels of immunoglobulins that may cause either agglutination or rouleaux
Hemolyzed red blood cells due to a difficult draw will interfere in your grading interpretation of hemolysis
Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.
Good Technique when washing and making a 3% cell suspension involves the following:
Place 1 to 3 drops of blood in the tube
Fill the tube 3/4 full of saline (there will be less splattering in the centrifuge)
Centrifuge long enough spin to pull most of cells into a button in the bottom of tube.
Decant the saline completely
Shake the tube to resuspend cell button before washing the cells again.
COLLECTING BLOOD BANK SAMPLES Samples for Blood Bank Testing
Most samples for blood banking are drawn into a red top tube - serum is preferred. (No clot activation tube should be used since the patient's red cells may also need to used and no other chemicals should be present)
A few tests require an EDTA sample if complement is not to be activated.
Serum must be tested while fresh to ensure good complement activity.
Antigens on cells are stable longer (months) in a clot tube.
QUALITY ASSURANCE IN BLOOD BANKING Quality Programs When discussing Quality Programs it includes:
Quality Improvement a. Responsibilities of the blood product requirements (anticoagulants and preservatives, shelf life etc.) b. Specific requirements related to independent quality control and quality assurance for overall quality of blood products and the processes related to dispersion of those products.
Organization: active support of quality systems must be place for the following procedures;
SOP's - Standard Operating Procedures
Training plans and development of procedures
Approval of lot release of reagents and quality control reagents
Review and approval of practices relating to personnel, equipment, selection of suppliers, process control, final inspection and handling nonconforming components, methods in place for handling incidents, errors, and accidents.
Validation of new equipment
Calibration and preventative maintenance including standard equipment like refrigerators, complex equipment and computer systems
Continual monitoring of blood bank refrigerators extremely important in both blood centers and transfusion services
Supplier issues Specific criteria is set for both the specificity and the potency of the reagents. For example, anti-A will only react with A cells and will demonstrate a 3-4+ reaction with A1 reagent cells. Other than very rare antisera, routine blood bank reagents CANNOT be used after the expiration date. Daily quality control testing needs to be done for ABO, Rh, and Antibody Screening. Typing antisera for other red cell antigens will be tested when performing the antigen testing on the patient and donors since this test is not done each day.
Each manufacturer is required to provide a product insert for each reagent. The product insert needs to include the following:
Proper use procedures
What to expect in regards to performance
When a new shipment of reagents is received, the product insert needs to be reviewed and any changes in the standard operating procedure. Total compliance with the manufacturer's directions must be followed. Process control, final inspection, and handling elements
Development of SOP
Control of changes in policies, processes or procedures
Acceptance testing to new/revised software involved in blood bank procedures
Validation of new policies, processes or procedures
Monitoring and control of production processes
Participation in proficiency testing appropriate for each testing system in place
Established QC procedures for supplies and equipment
Supplier qualifications and product specification need to be in place
Control processes for nonconforming blood and blood components and products.
Documents and records (4 levels) 1. Policies (Level 1) relate to "What to do" in response to various situations 2. Processes (Level 2) relate to "How it happens" 3. Procedures (Level 3) "How to do it" 4. Forms/Records, Supporting Documents etc. (Level 4) that need to be completed when you are performing the procedures and following the processes and policies. Incidents, errors, and accidents
If incident occurs, the severity of the incident is determined by the facility
If it is a one-time incident: "What is the likelihood it will happen again?" and what to do about it if it could happen again
If there are multiple similar incidents "What might be the root cause?"
Develop processes for continuous improvement to help eliminate both one-time incidents and multiple similar incidents.
Assessments: internal and external A Quality Assessment Program includes both internal and external assessment:
Internal assessment includes blood usage review committees within a hospital (transfusion audits) or institutional QA teams
External assessments includes inspections, surveys, proficiency surveys performed by agencies
Corrective actions that are educational not punitive
Yearly reports relating to QA and CQI committees
ANTIGLOBULIN TESTING The antiglobulin test, is to detect clinically significant unexpected antibodies that have coated cells either in vivo or in vitro. Principle of Antiglobulin Test Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to be sensitized with IgG or complement.
IgG-coated red cells
Complement-coated red cells
In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system. This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination.
The light-colored antibody molecule represents the anti-globulin reagent that binds with the complement attached to the red blood cells. Traditionally rabbits were immunized with human gamma globulin to make this antibody to IgG or C3d. Types of Antiglobulin Tests
Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo.
Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation step so antibodies in serum can react with antigens on cells in vitro. After washing the cells antiglobulin reagent is used to detect antibody coating of cells.
Reagents Production Methods of Anti-Human globulin (AHG or Coombs) Reagent
May be made by injecting rabbits with purified human IgG or C3, then harvesting the antibodies produced by the rabbit.
Monoclonal technology may be used to make monoclonal antiglobulin reagent
Specificity types Polyspecific Anti-human Globulin: blend of Anti-IgG & Anti-C3b, -C3d Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone Note: Reagent does not contain antibodies to IgM. Information about IgM coating of cells comes from the presence of C3 coating the cells since IgM is a strong complement activator.
Interpretation of Antiglobulin Tests Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following Positive Antiglobulin Test
Wash cells three times to remove unbound antibody Only antibody attached to the cells remain
Add Anti-Human Globulin
Summary of the reaction: 1. Antigen-antibody reaction, which can take place either in vivo or in vitro 2. Cells coated with IgG antibody and/or complement 3. Cells washed 3-4X to remove unbound or free antibody or complement 4. The only antibody or complement left is attached to red cells 5. AHG (Coombs serum) added 6. Antibodies in Coombs serum react with antibodies or complement on red cells, causing agglutination 7. If no agglutination add Coombs control reagent cells* (CCC).
Negative Antiglobulin Test
Antibodies are not attached to the antigens during incubation.
Wash the cells 3 times to remove any unattached antibodies. Add Anti-human globulin
No visible agglutination and therefore a negative test
Add Coombs Control Check Cells Check cells agglutinated and original test cells remain unagglutinated. Coombs Control Agglutinated by Anti-Human Globulin 1. NO antigen-antibody reaction occurred. 2. No attachment of antibody or complement to red cells 3. Cells washed three to four times = all plasma or serum antibodies was washed away. 4. Anti-human globulin, Coombs, serum added, which would react with antibody-coated cells if present. 5. But no agglutination, because no antibodies or complement on red cells for the anti-human globulin, Coombs, serum to react with 6. Must add Coombs Control Check Cells to negative reactions CCC are cells coated with IgG antibody Will react with antibodies in Coombs serum still "floating around" in the tube.
Agglutination will now result Agglutination following addition of CCC verifies negative result
False Positives and Negatives
Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody-coated cells
If the free antibody has already reacted with the anti-human globulin, no free Coombs serum to react with Coombs Control Check Cells (CCC)
False negatives that are detected by negative Coombs control cells includes: Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody. Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from, cell while cells are sitting in saline. Now free antibody present in the saline neutralizes AHG, Coombs, serum so it will not able to react with cells bound with antibody. Small fibrin clot among the cells that were not washed away will have immunoglobulins and complement present. The antibodies and complement in the fibrin clot neutralizes AHG, Coombs, serum leading to a negative test. Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be detected by a negative reaction when adding Coombs Control Check Cells. There are also false negatives NOT detected by negative Coombs Control Cells that include:
Too heavy cell suspension
Delay during cell washing procedure, which can lead to antibody eluting off cells while they are sitting in saline and then the antibody is washed away during the remaining washes
Improper centrifugation can either lead to lost of cells during the washing or the need to shake too hard during resuspension.
False positive reactions can also occurred when performing this test. These would not be detected by the use of Coombs Control Check Cells. Reasons for a false positive reaction could be the following:
Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT)
Spontaneous agglutination (cells heavily coated with IgM)
Non-specific agglutination ("sticky cells")
All of these reactions would be the result of cells appearing to agglutinate, or actually agglutinating. Using a clotted tube for the DAT may allow complement to become activated in the test tube since calcium ions are free to be part of the complement cascade.
Direct Antiglobulin Testing Principle The Direct Antiglobulin Test detects in vivo coating of patient cells - either IgG antibodies, complement, or both. Within the patient's blood stream antibodies attach to their specific antigens on the red blood cells. This happens in Hemolytic Disease of the Newborn (HDN), in transfusion reactions, and in autoimmune hemolytic anemia. Certain drugs are also known to activate complement and it can also coat the cells in vivo. When the blood is drawn the antibodies and/or complement have already attached to the red cells. Those red cells from the EDTA tube will be washed 3 or more times and a 3% cell suspension is made. A drop of cell suspension and the anti-human globulin are mixed in a tube and then centrifuged. If agglutination occurs, it indicates the patient has a positive Direct Antiglobulin Test due to antibody coating the cells in vivo. If IgM antibodies involved, DAT will be identified by complement binding since the polyspecific antisera has both anti-IgG and anti-C3. The meaning of a positive DAT is found under Clinical Causes of a Positive DAT.
1. Add 1 drop of patient cells from EDTA tube to tube 2. Wash 3-4X to remove plasma antibodies and make 3% cell suspension. 3. Add a drop of 3% cell suspension to a clean, labeled tube. 4. Add drop of Polyspecific AHG (Coombs serum) to the tube. 5. If test is positive with polyspecific reagent, set up again using monospecific reagents to see if it is antibody or complement or both coating the cells. 6. We want to make the test as sensitive as possible, so allow all negatives to incubate 5 minutes to enhance complement coating. 7. Read all negatives microscopically to detect weak coating. 8. False pos. possible if red top tube used to collect sample. In-vitro complement coating frequently happens when sample clots or cools down
due to weak cold-acting auto-antibodies like anti-I Prevent by using lavender top tube to tie up Ca+ and Mg+ ions and prevent
complement activation in vitro. 9. Whenever positive DAT is obtained, obtain the following information on the patient: Diagnosis (particularly autoimmune hemolytic anemia, HDN, transfusion reactions) Medications Recent transfusion history of both red cell and plasma components Other lab values that may indicate red cell destruction (hematocrit, bilirubin, LDH)
Clinical Causes of Positive DAT 1. Normal patient with unexplainable reasons for a positive DAT 2. Transfusion reaction work-ups require that a DAT be performed on the post-transfusion specimen since the patient's antibodies and/or complement may coat the transfused donor cells. These reactions are usually a weak positive or mixed field agglutination since you are testing a mixed population of patient and donor cells. 3. Warm-acting Autoimmune disease, can lead to patient antibodies coating their own cells. This results in a strong positive result. A cold-acting autoimmune hemolytic anemia would be due to IgM antibodies that in turn activate complement. The complement-coated cells would then be detected by the antiglobulin reagent. 4. Hemolytic disease of newborn is due to the mother's IgG antibodies crossing the placenta and coating the antigens on the fetal red cells. Cord blood collected at
time of birth would be tested, but may need to followed up by a heel stick of EDTA blood. The reaction is usually a strong positive. 5. Complement on the red cells may be the result of antigen-antibody reactions which may involve red cells. Complement can also be activated if immune complexes are present in the plasma and the activated complement attaches to red cells. Complement can also become activated by the C3 by-pass mechanism and the lectin activation process. Again once the activation of complement occurs in the blood stream, it can become attached to the red cells. 6. Passive transfer of antibody from donor units of plasma or platelets may attach to the patient's red cells since recipients are given ABO compatible blood but other unexpected red cell antibodies may not have been detected. These antibodies in donor plasma can coat antigens on patient cells when group AB, A, or B receive group O plasma products (and possibly platelets) 7. ABO mismatched transplants of particularly bone marrow can occur if an universal "O" donor bone marrow is given to an A, B, or AB recipient. "Passenger lymphocytes" from group O donor organ make antibody to group AB, A, or B recipient cells and these in turn can activate complement. It is also more common for "O" individuals to make an IgG anti-A,B, which would also contribute to a positive DAT. 8. Sensitization of red cells due to medications like penicillin and cephalosporins that usually involves non-specific coating of red cells. Other drugs like tetracyclines, antihistamines and sulphonamides cause the development of immune complexes that are capable of activating complement. Some drugs, like ibuproten, levodopa and methyldopa, are also known to cause autoimmunity. If a patient has a positive DAT, drug-induced problems should be considered. Indirect Antiglobulin Testing The indirect antiglobulin test is one of the most important and commonly used techniques in immunohematology. It is used to commonly for the detection of:
Weak D's in donor bloods and pregnant females of individuals who type D (-) at room temperature when doing ABO and Rh typing.
The presence or absence of antigens on a person cells from particularly the Kell, Kidd, and Duffy Blood Group systems.
Unexpected, clinically significant antibodies in the patient's serum during the antibody screening procedure and the antibody identification procedure
Principle The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells. This is done when sensitization does not lead to direct agglutination. This occurs when there are too few antigens on the red cell, too few antibodies in the serum and those antibodies are in the IgG class. Summary of the Indirect Antiglobulin Technique 1. Incubate cells with serum at 37oC for 15 to 30 minutes. 2. After incubation wash the cells three to four times. 3. Add AHG, Coombs reagent, centrifuge and read for agglutination. 4. If the test is negative, add Coombs Control Check Cells to check for false negatives. Screening Serum for Unexpected Antibodies Procedure 1. Involves patient serum plus reagent red cells (Screening Cells) The patient's serum potentially has unknown antibody. Screening Cells have known antigens for common clinically significant antibodies.
2. If there is agglutination after Coombs step with either (or both) Screening Cells, patient has an unexpected antibody. 3. If antibody screen positive, must do additional tests to specifically identify antibody The uses for antibody screen are:
Testing donor plasma to make sure no unexpected antibodies will be transfused to the recipient.
Testing recipient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells.
Testing maternal serum to make sure pregnant mother has no antibodies to react with fetal cells causing hemolytic disease of the newborn.
Red Cell Antigen Typing Red cell antigen typing involves patient cells plus reagent antiserum. The patient's cells are the unknown antigen and the reagent antiserum is the known antibody. The antiglobulin technique is used for antigen typing for a weak D and a number of other clinically significant antibodies like the Kell, Kidd, and Duffy antibodies. If there is agglutination after the addition of anti-human globulin, patient cells had that specific antigen.The specific procedure varies depending on what antigen is being tested for, and what brand of antiserum is being used. Uses for red cell antigen typing are:
Typing donors for antigen if patient has antibody.
Verifying that patient is negative for antigen if he/she has made the antibody.
Typing patient to see what antigens are lack, so can predict what antibodies is capable of making, if they seem to be likely to make additional antibodies. Controls
When performing red cell antigen testing always run known positive and negative controls. This will verify that antiserum is acting properly. The positive control should be heterozygous for the antigen to ensure antiserum is capable of detecting weaker antigens. For example, when performing antigen typing for K, you would want a cell that is K+ and k+.
Cells without Antigen
0 / MF
Heterozygous Positive cells
May be Positive (2+) Should be at least 2+
Should be Negative
Should be Negative
Negative or Mixed Field
GENETICS IN BLOOD BANKING Mendelian Inheritance and Significance Terms Basic Principles: 1. Each parent contributes 1/2 of the genetic information. 2. The genetic information is contained on chromosomes composed of DNA 3. Humans have 23 pairs of chromosomes a. 22 matched (autosomal) chromosomes and b. 1pair of sex chromosomes (females have 2X and males XY). 4. Examples of Chromosome locations for common Blood Groups are as follows: System
Located on Chromosome
A, B, O
M, N, S,s,U
D, C, E, c, e
K, k, Kpa, Jsa,Kpb, Jsb
Fya, Fyb, Fy3
5. Genes are the units of inheritance within the chromosomes. 6. At each location, or loci, on the chromosomes there are possibilities of different forms of the genes, these different forms are called alleles. (For example ABO Blood Group System, there are A1, A2, B, and O as common alleles. or allelic genes) 7. When the inherited alleles are the same the person is homozygous such as OO, when the individual inherits 2 different alleles such as AO, they are heterozygous for both A and O genes. 8. On occasion we will see examples of dosage where some antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E that reacts as a 3+ with EE cells and only 1+ with Ee cells. 9. A Punnett Square is used to determine the inheritance possibilities for a particular mating. For example if the mother's genotype (genes) are AO and the father's genotype (genes) are BO. In this example there three heterozygous possibilities AB, AO, and BO and one homozygous possibility OO
10. In the above Punnett Square, the AB genotype will have both A and B antigens, therefore the phenotype is AB since both are expressed. AO and BO genotypes will demonstrate only the A and the B antigens respectively and therefore the phenotypes are A and B respectively. The individual that is OO will have the O phenotype. 11. A and B genes are dominant, or co-dominant, and the O gene is recessive. The dominant genes will be expressed if present. Recessive genes will only be expressed if they are homozygous. 12. Most Blood Group genes are co-dominant and therefore will be expressed if present. Mitosis and Meiosis Two kinds of cell division: Mitosis is cell division that leads to two identical cells that has the same number of paired chromosomes. (In humans there are 23 pairs or 46 chromosomes) Meiosis is the cell division that occurs when gametes (sperm and eggs) are formed and will not have pairs of chromosomes. (In humans there will be 23 chromosomes in the sperm that will match up with the 23 chromosomes in the egg when fertilization occurs to form the gametocyte.). The sex of the child is determined by the X and Y chromosomes. Males provide either X or Y chromosome and females provide only provide X chromosomes. Genes that are found only on the X chromosome are said to be sex-linked. Genes found on the other 22 pairs of chromosomes are autosomal. Genotypes, Phenotypes, Amorphs, and Pedigree Charts Here is a pedigree chart for three generations. The ABO phenotypes are listed for the known blood types.
The mother in the first generation has the AB genes since her phenotype is AB.
In the mating for the second generation, the genotype for the father could either be BB or BO
since his father's phenotype is unknown. It would appear the mother is AA since both her parents are A, but.....
There are no O individuals in above example but O is considered an amorph since it has no detectible traits. The lack of D antigen is also considered as an amorph since no reaction with anti-D indicates the individual is D negative. These two examples are recessive genes that need to be homozygous for it to be demonstrated.
Other Concepts Relating to Blood Group Genetics Contributions of Blood Genetics to the Field of Human Genetics Certain characteristics that make Blood Genetics useful for the field of human genetics 1. Simple and unquestionable pattern of inheritance 2. Can test or determine the phenotypes readily 3. More than 1 allele occurring fairly frequently 4. Environment does not affect the expression of the genes. Some discoveries that were found in blood genetics:
Multiple alleles seen in ABO system
Population Genetics Linkage Linkage between the secretor genes with the Lutheran genes on the same chromosome was already noted. 1. We now know that the D gene is closely linked to the Cc and Ee genes. The most frequently inherited Rh positive set of genes is CDe and the most frequent Rh negative gene is cde or ce since d is an amorph. 2. The MNSs genes are also linked, MS, NS, Ms, Ns leading to a difference between the expected frequency and the observed frequency. Expected frequency
MS = 0.53 (M) X 0.33 (S) = 0.17
Ms = 0.53 (M) X 0.67 (s) = 0.36
NS = 0.47 (N) X 0.33 (S) = 0.16
Ns = 0.47 (N) X 0.67 (S) = 0.31
Silent Genes As indicated already there are some amorph blood group genes that exist and lead to none expression of a blood antigen. The following are some examples of silent genes. Blood Group Gene
Blood Group System
Oh or Bombay
Public versus Private Genes
Public Genes are found in most of the population. In the Kell Blood Group System, the Kpb is found in close to 100% of the population
Genes that are very rare are referred to private genes. Kpa is very rarely found (2.3% in whites and almost never in African Americans).
Paternity Testing Today most paternity testing is done using the following technology:
Red Cell Testing for: ABO, MNSs, Rh, Duffy, Kidd, Kell
White Cell Testing using HLA antigens
ABO BLOOD GROUP SYSTEM Blood group system A series of antigens exhibiting similar serological and physiological characteristics, and inherited according to a specific pattern. Importance of the ABO system Most important (clinically significant) Blood Group System for transfusion practice Why? This is the only blood group system in which antibodies are consistently, predictably, and naturally present in serum of people who lack the antigen. Therefore ABO compatibility between donor and recipient is crucial since these strong, naturally occurring A and B antibodies are IgM and can readily activate complement and cause agglutination. If ABO antibodies react with antigens in vivo, result is acute hemolysis and possibly death. Indications for ABO grouping: ABO grouping is required for all of the following individuals:
Blood Donors Since it can be life threatening to give wrong ABO group to patient.
Transfusion recipients Since we need to know the donor blood is ABO compatible.
Transplant Candidates and Donors ABO antigens are found in other tissues as well. Therefore the transplant candidates and donors must be compatible.
Prenatal Patients To determine whether mothers may have babies who are suffering from ABO-HDN.
Newborns (sometimes) If the baby is demonstrating symptoms of Hemolytic Disease of Newborn, the ABO group needs to be determined along with Rh and others.
Paternity testing Since the inheritance of ABO Blood Group System is very specific, this serves as method to determine the likelihood the accused father is the father or not.
Landsteiner's rules for the ABO Blood Group 1. A person does not have antibody to his own antigens 2. Each person has antibody to the antigen he lacks (only in the ABO system) 3. Below are the four blood groups and the antigens and the expected, naturally-occurring antibodies present.
ABO Typing ABO typing involves both antigen typing and antibody detection. The antigen typing is referred to as the forward typing and the antibody detection is the reverse typing
The forward typing determines antigens on patient's or donor's cells a. Cells are tested with antisera reagents anti-A, anti-B, (donor cells anti-A,B) b. Reagents are made from monoclonal antibodies. c. One advantage of the monoclonal antibodies are the antibody strength. d. Another advantage of monoclonals: human source reagents can transmit infectious disease (hepatitis).
Reverse typing determines antibodies in patient's or donor's serum or plasma a. Serum tested with reagent A1 cells and B cells b. Reverse grouping is also known as backtyping or serum confirmation Routine ABO Typing
Reaction of Cells Tested With
Reaction of Serum Tested Against
Reverse ABO Group
Discrepancies in ABO typing 1. Results of forward and reverse typing must agree before reporting 2. If forward and reverse do not agree, must identify cause of discrepancy 3. If cannot resolve discrepancy, must report blood type as UNKNOWN and give group O blood Characteristics of ABO antigens ABO antigens are glycolipid in nature, which are oligosaccharides attached directly to lipids on red cell membrane. These antigens stick out from red cell membrane and there are many antigen sites per red blood cell. Besides the presence on red cells, soluble antigens can be present in plasma, saliva, and other secretions. These antigens are also expressed on tissues other than red cells. ABO antigens are only moderately well developed at birth. Therefore ABO-HDN not as severe as other kinds.
Characteristics of ABO antibodies 1. These are expected naturally occurring antibodies occur without exposure to red cells containing the antigen. 2. IgM antibodies, predominantly 3. They react in saline and readily agglutinate. Due to the position of the antigen and the IgM antibodies it is not necessary to overcome the zeta potential. 4. Optimum temperature is less than 30oC, but reactions take place at body temp 5. Not only are these antibodies expected and naturally occurring, they are also commonly present in high titer, 1/128 or 1/256. 6. They are absent at birth and start to appear around 3-6 months as result of stimulus by bacterial polysaccharides. (For this reason, newborn blood is only forward typed.) ABO INHERITANCE gene: determines specific inherited trait (ex. blood type) chromosome: unit of inheritance. Carries genes. 23 pairs of chromosomes per person, carrying many genes. One chromosome inherited from mother, one from father locus: site on chromosome where specific gene is located allele: alternate choice of genes at a locus (ex. A or B; C or c, Lewis a or Lewis b) homozygous: alleles are the same for any given trait on both chromosome (ex. A/A) heterozygous: alleles for a given trait are different on each chromosome (ex. A/B or A/O) phenotype: observed inherited trait (ex. group A or Rh positive) genotype: actual genetic information for a trait carried on each chromosome (ex. O/O or A/O) dominant: the expressed characteristic on one chromosome takes precedence over the characteristic determined on the other chromosome (ex. A/O types as A) co-dominant: the characteristics determined by the genes on both chromosomes are both expressed - neither is dominant over the other (ex. A/B types as AB) recessive: the characteristic determined by the allele will only be expressed if the same allele is on the other chromosome also (ex. can type as O only when genotype is O/O)
ABO Genes The A and B genes found on chromosome 9. We inherit one gene (allele) from our father and one from our mother. The two co-dominant alleles are A or B. Anytime an individual inherits an A or B gene it will be expressed. The O gene is not expressed unless this gene is inherited from both parents (OO). Therefore O gene is recessive. Below is example of two individuals who are A. One inherited only one A gene along with an O gene and is therefore heterozygous. The other inherited 2A genes and is homozygous.
1 = A/A
2 = A/O
Can Contribute Only an A Gene to Offspring
Can Contribute A or O Gene to Offspring
Inheritance Patterns 1. A/A parent can only pass along A gene 2. A/O parent can pass along either A or O gene 3. B/B parent can only pass along B gene 4. B/O parent can pass along either B or O gene 5. O/O parent can only pass along O gene 6. AB parent can pass along either A or B gene ABO phenotypes and genotypes 1. 1. Group A phenotype = A/A or A/O genotype 2. 2. Group B phenotype = B/B or B/O genotype 3. 3. Group O phenotype = O/O genotype 4. 4. Group AB phenotype = A/B genotype
BIOCHEMISTRY OF THE ABO SYSTEM The ABO antigens are terminal sugars found at the end of long sugar chains (oligosaccharides) that are attached to lipids on the red cell membrane. The A and B antigens are the last sugar added to the chain. The "O" antigen is the lack of A or B antigens but it does have the most amount of next to last terminal sugar that is called the H antigen.
Production of A, B, and H antigens The production of A, B and H antigens are controlled by the action of transferases. These transferases are enzymes that catalyze addition of specific sugars to the oligosaccharide chain. The H, A, or B genes each produce a different transferase, which adds a different specific sugar to the oligosaccharide chain. 1. Precursor chain of sugars is formed most frequently as either Type 1 or 2 depending on linkage site between N-acetylglucosamine (G1cNAc) & Galactose (Gal). 2. H gene causes L-fucose to be added to terminal sugar of precursor chain, producing H antigen (Type 2 H antigen saccharide chaine).
Either A gene causes N-acetyl-galactosamine to be added to H substance, producing A antigen, (shown in this diagram) or
4. B gene causes D-galactose to be added to H substance, producing B antigen.
5. If both A and B genes present, some H-chains converted to A antigen, some converted to B antigen. 6. If H gene absent (extremely rare), no H substance can be formed, and therefore no A or B antigen. Result is Bombay blood group.
Bombay blood group Bombay blood group lacks H gene and therefore cannot make H antigen (H substance). Since the H substance is the precursor for the A and B antigens, these antigens also are not made. The cells type as O and the serum has anti-A, anti-B, and anti-H since the individual lacks all of these antigens. Anti-H agglutinates O cells. The only cells Bombay individuals do not agglutinate are from other Bombay blood people since they lack H antigen, Bombay phenotype
H antigen is not expressed on
H antigen is weakly expressed on RBCs.
H antigen may be present or absent in
H antigen is not found in saliva.
Serum contains anti-H.
Serum contains anti-H.
Genotype: h/h se/se
Genotype: (H), Se/Se or Se/se or se/se
Subgroups of A and B The subgroups of A and B are caused by decreased amounts of antigen on red cells. The most common are subgroups of A. Approximately 80% of the A's and AB's have a normal expression of A1. Most of the other 20% are either A2 or A2B. This subgroup has fewer H chains converted to A antigen – result is more H chains on red cell, and fewer A antigens. There are other, weaker subgroups of A exist: A3; Aint; Am, Ax; Ael. Each has a different pattern of reacting with anti-A, anti-A, and various antibody-like substances called lectins. Lectins are extracts of seeds of plants that react specifically with certain antigens.
Ulex europaeus, or lectin H, which agglutinates cells that have H substance.
Dolichos biflouros, or lectin A1, which agglutinates cells with A1.
Lectin-H reacts strongest with O cells, which has a high concentration of H antigen, and weakest with A1 cells, which have a low concentration of H. Bombay
1+ / 0
1+ / 0
Differentiating Subgroups of A 1. Use lectin-A1 to differentiate A1 cells from all others - will agglutinate only A1 cells 2. Look for weaker or mixed field reactions 3. Look for anti-A1 in serum (serum reacts with A1 cells but not A2 cells) 4. Look at strength of reactions with anti-A,B or with lectin-H GROUP
Reaction with anti-A
Reaction with anti-A,B
Reaction with Lectin-A1
Reaction with Lectin-H
Presence of anti-A1
often in serum
Problems with Ax: Because Ax cells initially type as O and serum usually has anti-A1, (along with anti-B), patient forwards and reverses as O. Unfortunately when Ax is transfused into an O individual, the naturally occurring anti-A,B will react with the donor cells causing a transfusion reaction. Therefore: To prevent Ax from being erroneously typed as O, confirm all group O donors with anti-A,B. Rh SYSTEM Rh Antigens D (Rh) is the most important antigen after A and B antigens. Unlike the anti-A and anti-B antibodies, anti-D antibodies are only seen if a patient lacking D antigen is exposed to D+ cells. The exposure of D+ cells usually occurs through pregnancy or transfusion. There are at over 50 Rh antigens that have been identified including those that are either combinations of these antigens or weak expressions of the above antigens, but most Rh problems are due to D, C, E, c or e. Characteristics of Rh antigens The Rh antigens together are proteins of 417 amino acids. These proteins cross the red cell membrane 12 times. There are only small loops of the protein on the exterior of the cell membrane.
Therefore Rh antigens are not as available to react with their specific antibodies and there are fewer antigen sites than ABO. Unlike the ABO system, Rh antigens are not soluble and are not expressed on tissues. They are well developed at birth and therefore can easily cause hemolytic disease of the newborn if the baby has a Rh antigen that the mother lacks. Besides the antigens being well-developed at birth, they are very good immunogens. This is especially true to D, which if the most immunogenic after A and B antigens.
Rh Antibodies Unlike the ABO antibodies that are mainly IgM, Rh antibodies are commonly IgG. They are NOT naturally occurring and therefore are formed by immune stimulus due to transfusions or baby's red blood cells during pregnancy. The most common antibody to form is anti-D in Rh negative individuals. Since Rh antibodies are IgG they bind best at 37oC and their reactions will be observed with indirect antiglobulin technique. Agglutination reactions are enhanced by high protein (albumin), low-ionic strength saline (LISS), proteolytic enzymes (papain) and polytheylene glycol (PEG). Rh antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E will react with strongly with E+E+ cells and more weakly with E+e+ cells. This is called dosage effect. Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions can occur due the various Rh antibodies. Anti-D is the most common cause of hemolytic disease of the newborn. Since D antigen is so immunogenic we screen all donor units for the D antigen. Therefore if an individual is A+, it means both the A and the D antigens are present. On the other hand, if an individual is A-, the A antigen is present and the D antigen is absent. To prevent problems due to anti-D:
always give Rh-negative individuals Rh-negative blood
give Rh immune globulin to Rh-negative mothers to prevent formation of anti-D during pregnancy.
The incidence of Rh antibodies Anti-D most common antibody seen in Rh(D) negative people Anti-E most common antibody seen in Rh pos people since only 30% of population have
the antigen Anti-C or Anti-c less common - most people have the antigen Anti-e often seen as autoantibody and will make it difficult to find compatible blood
since 98% of the population have the e antigen Anti-C,e or Anti-c,E often seen in combination. If a patient lacks both a C and e and
has made an anti-C, then enhancement techniques should be done to make sure that an anti-e is not also present.
Rh System Inheritance Fisher-Race theory involved the presence of 3 separate genes D, C, and E and their alleles c and e and the absence of D since an anti-d has never been found. These three genes are closely linked on the same chromosome and are inherited as a group of 3. The most common group of 3 genes inherited is CDe and ced is the second most common. Weiner Theory Weiner believed there was one gene complex with a number of alleles resulting in the presence of various Rh antigens. According to Weiner there were 8 alleles, R, R1, R2, Rz, r, r', r", ry , which ended up with different antigens on the red cells that he called Rh, rh', rh", hr', hr". If a person has the Fisher-Race genotype of DCe/DCe, it is easier to refer to that type as R1R1 Tippett Theory Tippett predicted that there are two closely-linked genes - RHD and RHCE. The RHD gene determines whether the D antigen that spans the membrane is present. Caucasians who are D negative have no gene at that gene loci. The RHCE gene determines C, c, E, e antigens produced from the alleles:
RHce Rh Gene Complexes, Antigens, Possible Combinations and Percentages